Systems and methods for evaluating target molecules

Abstract

This disclosure provides systems and methods for analyzing or identifying target molecules. [Solution] For example, the system of the present disclosure may include (i) a sensing electrode, (ii) a bonding unit coupled to the sensing electrode and configured to bond to at least a portion of a target molecule, and (iii) a dielectric material coupled to the sensing electrode and covering at least a portion of the surface of the sensing electrode. The sensor may be configured to detect one or more signals indicating the impedance of the sensor or an impedance change when at least a portion of the target molecule is bonded by the bonding unit. One or more signals may be used to analyze or identify the target molecule.

Description

[Technical Field]

[0001] cross reference

[0001] This application claims the benefits of U.S. Provisional Patent Application No. 62 / 881,254, filed on 31 July 2019, which is incorporated herein by reference in whole. [Background technology]

[0002] background

[0002] Nucleic acid sequencing can be used to provide sequence information of nucleic acid samples. Such sequence information can be useful in diagnosing or treating a condition (e.g., disease) of a subject (e.g., an individual, a patient, etc.). For example, nucleic acid sequence information of a subject can be used to identify, diagnose, or develop treatments for one or more genetic diseases. In another example, nucleic acid sequence information of one or more pathogens can lead to the treatment of 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 pathogenic mutations and for their early detection. The detection of one or more cancer-related 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 patients with relapse. In addition, such detection of one or more mutations may be important for evaluating exposure to environmental mutagens, monitoring endogenous DNA repair, or studying 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 enable the characterization of fetal cells present in maternal blood.

[0004]

[0004] Alternatively, or in addition, the detection of small molecules and / or polypeptides (e.g., growth factors, enzymes, etc.) from biological samples (e.g., blood, urine, tissue biopsy, etc.) may be used to identify or monitor one or more health conditions, such as cancer. [Overview of the project] [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 comprising 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 a target molecule, and (iii) a dielectric material coupled to the sensing electrode and covering at least a portion of the surface of the sensing electrode, the sensor being configured to detect one or more signals indicating the impedance or 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 for analyzing or identifying the target molecule.

[0006]

[0006] In some embodiments, one or more signals indicate one or more members including (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]

[0007] In any one partial embodiment of the subject system, one or more signals are currents or voltages. In any one partial embodiment of the subject system, one or more signals are not tunnel currents.

[0008]

[0008] In some embodiments of any one of the subject systems, the average cross-sectional dimensions of the sensing electrodes are 20 times or less the average size of the target molecule. In some embodiments, the average cross-sectional dimensions of the sensing electrodes are 2 times or less the average size of the target molecule.

[0009]

[0009] In any one of the embodiments of the subject system, the average cross-sectional dimensions of the sensing electrode are smaller than the average size of the target molecule.

[0010]

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

[0011]

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

[0012]

[0012] In any one of the embodiments of the subject system, the target molecule includes a tag, which is configured to induce changes in one or more signals.

[0013]

[0013] In some embodiments of any one of the systems of the subject, the sensor further includes a reference electrode electrically in communication with a sensing electrode, and one or more signals indicate an impedance or 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 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]

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

[0015]

[0015] In any one of the embodiments of the subject system, the binding unit comprises one or more members selected from the group consisting of small molecules, enzymes, antibodies, functional fragments thereof and functional variants thereof.

[0016]

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

[0017]

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

[0018]

[0018] Different aspects of the present disclosure provide a method for analyzing or identifying a target molecule, the method 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 a target molecule; and (iii) a dielectric material coupled to the sensing electrode and covering at least a portion of the surface of the sensing electrode; (b) detecting one or more signals indicating the 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 a target molecule using one or more signals.

[0019]

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

[0020]

[0020] In any one partial embodiment of the subject method, one or more signals are currents or voltages. In any one partial embodiment of the subject method, one or more signals are not tunnel currents.

[0021]

[0021] In some embodiments of any one of the methods of the subject, the average cross-sectional dimensions of the sensing electrodes are 20 times or less the average size of the target molecules. In some embodiments, the average cross-sectional dimensions of the sensing electrodes are 2 times or less the average size of the target molecules.

[0022]

[0022] In any one of the embodiments of the subject method, the average cross-sectional dimensions of the sensing electrode are smaller than the average size of the target molecule.

[0023]

[0023] In any one part of the method of the subject, the coupling unit is coupled to the sensing electrode via a conductive material.

[0024]

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

[0025]

[0025] In any one of the embodiments of the subject method, the target molecule includes a tag, which is configured to induce changes in one or more signals.

[0026]

[0026] In some embodiments of any one of the methods of the subject, the sensor further includes a reference electrode electrically in communication with a sensing electrode, and one or more signals indicate an impedance or 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 using an additional field generator 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.

[0027]

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

[0028]

[0028] In any one sub-embodied embodiment of the subject method, the binding unit comprises one or more members selected from the group consisting of small molecules, enzymes, antibodies, functional fragments thereof and functional variants thereof.

[0029]

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

[0030]

[0030] Further aspects and advantages of the Disclosure will be readily apparent to those skilled in the art from the following detailed description, which illustrates and describes only exemplary embodiments of the Disclosure. As will be recognized, other different embodiments of the Disclosure are possible, and some of its details can be improved in various obvious ways without departing from the Disclosure. Accordingly, the drawings and description should be considered exemplary and not limiting.

[0031] Reference

[0031] All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference. To the extent that any publications, patents, or patent applications incorporated by reference conflict with the disclosure contained herein, this Specified Version is intended to take precedence and / or supersede any such conflicting material.

[0032] Brief explanation of the drawing

[0032] Novel features of the present invention are described in detail in the appended claims. Further understanding of the features and advantages of the present invention will be gained by referring to the following detailed description and the appended drawings (also referred to herein as "Figure" and "FIG.") illustrating exemplary embodiments in which the principles of the present invention are utilized. [Brief explanation of the drawing]

[0033] [Figure 1A]

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

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

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

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

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

[0034] Another example of a sensor for analyzing or identifying target molecules is schematically illustrated. [Figure 2B]

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

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

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

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

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

[0036] A schematic diagram shows an example of a sensor with an additional field generator. [Figure 4B]

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

[0037] A schematic diagram shows 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 schematically illustrated. [Figure 6]

[0038] This refers to a computer system programmed or otherwise configured to implement the methods provided herein. [Figure 7]

[0039] This section illustrates an exemplary process for analyzing or identifying target molecules. [Modes for carrying out the invention]

[0034] Detailed explanation

[0040] Various embodiments of the present invention are shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided merely as examples. Those skilled in the art will also be able to conceive of numerous variations, modifications, and substitutions without departing from the present invention. It should be understood that various alternative forms may be used instead of the embodiments of the present invention described herein.

[0035]

[0041] When used herein and in the claims, the singular form "one (a)" The words "an" and "that" can refer to plurals unless explicitly indicated otherwise in the context. For example, the term "transmembrane receptor" can refer to multiple transmembrane receptors.

[0036]

[0042] As used herein, the terms “about” or “approximately” may mean that a particular value is within an acceptable margin of error, as determined by those skilled in the art, and that the acceptable margin of error will, in part, depend on how the value is measured or determined, i.e., the limits of the measurement system. For example, “about” may mean that, according to the convention of the art, a value is within one standard deviation or greater than one standard deviation. Alternatively, “about” may mean 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 a range of a certain number of decimal places of a value, preferably within five times, more preferably within two times. Where a particular value is described in this application and claims, unless otherwise specified, the term “about” should be assumed to mean that the value is within an acceptable margin of error.

[0037]

[0043] As used herein, the term "dielectric material" 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, electric charge may cease to flow through it. In some cases, the dielectric material may exhibit dielectric polarization in such an electric field, where positive charges may move along the electric field and negative charges may move in the opposite direction, thereby creating an internal electric field that can partially cancel out the external electric field within the dielectric material. Examples of dielectric materials, though not limited to them, include polyester, polyethylene, polypropylene, fabric (such as nylon), paper, laminates, glass, and self-assembled monolayers (SAMs).

[0038]

[0044] The terms “self-assembled monolayer” and “SAM,” as used synonymously herein, refer to an aggregate (e.g., one or more layers) of molecules adsorbed in an ordered or relatively ordered manner on a surface (e.g., the surface of an electrode). Multiple molecules within a SAM may be oriented substantially parallel to one another. Multiple molecules within a SAM may be substantially perpendicular to the surface. Each molecule may include a functional group configured to adhere to the surface and a portion configured to interact with one or more neighboring molecules in the SAM (e.g., through hydrophobic interactions) to form a relatively ordered arrangement. The functional group may be configured to bond covalently or acovalently to the surface. In some examples, the functional group may be a thiol covalently coupled to a metallic surface, such as a gold surface. Depending on the circumstances, a SAM may include a single molecular aggregate or a mixed molecular aggregate. A mixed molecular aggregate may exhibit (expose) multiple additional functionalities (e.g., biological functionalities, bonding moieties, etc.). In some embodiments, SAMs can be used as sensors (e.g., biosensors, chemical sensors, etc.) to detect molecules (e.g., small molecules, biological molecules, etc.) in samples such as biological samples. Examples of SAMs, but not limited to, include 3-mercaptopropyltrimethoxysilane (3MPT), 3-aminopropyltrimethoxysilane (APTES), p-aminophenyltrimethoxysilane (APTS), and 4-[2-(triethoxysilyl)ethyl]pyridine. In some cases, one or more conductive materials may be present within the SAM and in contact with the surface.

[0039]

[0045] As used herein, the term “conductive material” may refer to any material capable of conducting electric current, including metals (e.g., tungsten, titanium, tantalum, aluminum, copper) and nonmetals (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] As used herein, the term “binding unit” generally refers to a molecule having the ability to interact with (e.g., bind to) one or more target molecules. Such binding may 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 may be reversible or irreversible. Alternatively, or in addition, the binding unit may be configured to bind to a tag (or probe) coupled to the target molecule. The target molecule may be a biomolecule. The target molecule may be a cell or one or more components of a cell or derivatives thereof.

[0041]

[0047] As used herein, the term “biomolecule” may refer to any molecule, its derivatives, or its functional variants found in a living system. Biomolecules may be naturally occurring or the result of external disturbances to a system (e.g., disease, poisoning, genetic engineering), and may also be synthetic analogs and derivatives thereof. Non-limiting examples of biomolecules 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. Biomolecules may be isolated from natural sources, or they may be synthetic.

[0042]

[0048] As used herein, the term “cell” generally refers to a biological cell or cell derivative. A cell can be the basic structural, functional, and / or biological unit of an organism. A cell can originate from any organism that has 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, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, liverworts, mosses), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Na (Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, etc.), Examples 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), and cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.). Sometimes, cells are not derived from naturally occurring organisms (for example, cells may be synthetically created cells, sometimes called artificial cells).

[0043]

[0049] The terms “nucleotide,” “nucleic acid base,” and “base,” as used synonymously herein, generally refer to a base-sugar-phosphate combination. Nucleotides may include synthetic nucleotides. Nucleotides may include synthetic nucleotide analogs. Nucleotides can be monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). Nucleotides in the term include ribonucleoside triphosphates such as adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), uridine triphosphate (UTP), and deoxyribonucleic acid. The nucleotides may include 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 containing these. As used herein, the term nucleotide generally refers to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Exemplary examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may be unlabeled or may be labeled with detectable labels. Labeling may also be performed using quantum dots. Detectable labels may include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzymatic labels. Examples of 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, and [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 are available from Heights, Ill. Cy3-dUTP and FluoroLink Cy5-dUTP; Fluorescein-15-dATP and Fluorescein-12- available from Boehringer Mannheim, Indianapolis, India. dUTP, tetramethylrhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP and fluorescein-15-2'-dATP; and chromosome labels available from Molecular Probes, Eugene, Oreg. Nucleotides can be listed as 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. A chemically modified single nucleotide may be biotin-dNTP. Some non-exclusive 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] Naturally occurring nucleotides, guanine, cytosine, adenine, thymine, and uracil can be abbreviated as G, C, A, T, and U, respectively. Nucleotides may contain any subunits that can be incorporated into a growing nucleic acid chain. Such subunits may be specific to A, C, G, T, or U, or one or more complementary A, C, G, T, or U, or any other subunit complementary to a purine (i.e., A or G or its variant) or pyrimidine (i.e., C, T, or U or its variant). This can enable the degradation of individual nucleic acid bases 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 synonymously herein, generally refer to nucleotides (either deoxyribonucleotides or ribonucleotides) or analogues in polymer form of any length, in single-stranded, double-stranded, or multi-stranded form. Polynucleotides may be exogenous or endogenous to cells. Polynucleotides may exist in a cell-free environment. Polynucleotides may be genes or fragments thereof. Polynucleotides may be DNA. Polynucleotides may be RNA. Polynucleotides may have any three-dimensional structure and may perform any function. Polynucleotides may contain one or more analogues (e.g., modified backbone, sugar, or nucleic acid base). Modifications to the nucleotide structure, if present, may be conferred before or after the assembly of the polymer. Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acids, xeno nucleic acids, morpholino, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein coupled to sugars), thiol-containing nucleotides, biotin-coupled nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, cuosin, and waiosin.Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, one or more loci defined by 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 a nucleotide may be interrupted by non-nucleotide components. A polynucleotide may contain 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 polymer assembly. The nucleotide sequence may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation with labeling components.

[0046]

[0052] As used herein, the term “gene” generally refers to the corresponding nucleotide sequence involved in encoding nucleic acids (e.g., DNA such as genomic DNA and cDNA) and RNA transcripts. As used herein in relation to genomic DNA, the term “gene” may include intervening non-coding and regulatory regions, and may include the 5' and 3' ends. In some usages, the term encompasses the transcription sequence, including the 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcription region will contain an “open reading frame” that codes for a polypeptide. In some usages of this term, “gene” includes only the coding sequence necessary to code for a polypeptide (e.g., the “open reading frame” or “coding region”). Genes may not code for polypeptides, such as ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. The term “gene” may include not only the transcription sequence but also non-transcription regions, including upstream and downstream regulatory regions, enhancers, and promoters. Genes can be “endogenous genes” or native genes, located in their natural position within the genome of an organism. Genes can also be “exogenous genes.” These may be "offspring" or non-natural genes. Non-natural genes are genes that are not normally found in their host organism but are introduced into the host organism through gene transfer (e.g., trans genes). Non-natural genes may be naturally occurring nucleic acids or polypeptide sequences that contain mutations, insertions and / or deletions (e.g., non-natural sequences).

[0047]

[0053] As used herein, the term “mutation” generally refers to a change in the nucleotide sequence in a normally conserved nucleic acid sequence, resulting in the formation of a mutant that is distinguishable from the normal (unmodified) sequence or wild-type sequence. The location (e.g., relative to the gene or sample polynucleotide) and sequence of the mutation may not be determined before sequencing. Alternatively, the location (e.g., relative to the gene or sample polynucleotide) and sequence of the mutation may be determined before sequencing, in which case sequencing may be performed to detect the presence or absence of mutations 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] As used herein, the term “probe” generally refers to a nucleotide or polynucleotide tagged with a marker (e.g., a fluorescent marker) useful for detecting or identifying the corresponding target nucleotide or polynucleotide by hybridization with the corresponding target sequence in a hybridization reaction. As used herein synonymously, the terms “nucleotide probe,” “nucleotide tag,” and “tagged nucleotide” generally refer to a probe having a single nucleotide. As used herein synonymously, the terms “polynucleotide probe,” “polynucleotide tag,” and “tagged polynucleotide” 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 perfectly complementary to one or more target polynucleotides in a sample, or it may contain one or more nucleotides that are not complementary to one or more nucleotides of one or more target polynucleotides in a sample (i.e., a mismatch).

[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 or functional group portion of a molecule or compound) that can be oxidized and / or reduced (i.e., “redox”) or undergo a Faraday reaction upon or in response to an electrical stimulus (e.g., upon or in response to an applied electric potential). In some examples, a redox species may comprise one or more molecular portions that accept and / or donate one or more electrons depending on their redox state. Depending on the case, a redox species may form a small molecule, a compound, a portion of a polymer molecule (e.g., a molecular portion), or 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), their derivatives, their functional variants, and combinations thereof. Examples of Fc derivatives include methylferrocene, dimethylferrocene, ethylferrocene, propylferrocene, n-butylferrocene, t-butylferrocene, and 1,1-dicarboxylic acid ferrocene.

[0050]

[0056] The same nucleotide can be tagged with the same marker. Conversely, the same nucleotide can 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 and second markers are different. If a sensor can detect and distinguish the first and second markers from each other separately, using multiple markers on the same nucleotide may help in solving the sequencing of identical nucleotides provided sequentially.

[0051]

[0057] The terms “complement,” “multiple complements,” “complementary,” and “complementarity,” as used synonymously herein, generally refer to sequences that are fully complementary to a given sequence and capable of hybridizing to it. A sequence that hybridizes to a given nucleic acid is called a “complement” or “reverse complement” of that given molecule if its base sequence over a given region has the ability to bind complementaryally to the base sequence of its binding partner, for example, to form AT, AU, GC, and GU base pairs. Generally, a first sequence capable of hybridizing to a second sequence is capable of hybridizing to the second sequence specifically or selectively, i.e., hybridization to the second sequence or a set of second sequences is preferred over hybridization to a non-target sequence during the hybridization reaction (e.g., it is more thermodynamically stable under a given set of conditions, such as stringent conditions commonly used in the art). Typically, hybridizable sequences share some degree of sequence complementarity over all or part of their respective lengths, including 25% to 100% complementarity, with 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 contain 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 nucleotides or more.

[0052] Sequence identity is not limited to the purpose of evaluating percent complementarity, but can be used with the Needleman-Bunsch algorithm (see, for example, the EMBOSS Needle aligner available at www.ebi.ac.uk / Tools / psa / emboss_needle / nucleotide.html (optional, with default settings)) or the BLAST algorithm (see, for example, blast.ncbi.nlm.nih.gov / Blast.cgi). Refer to the available BLAST alignment tools (optional selection, default settings applied). (i) or any suitable alignment algorithm, including the Smith-Waterman algorithm (see, for example, the EMBOSS Water Aligner available at www.ebi.ac.uk / Tools / psa / emboss_water / nucleotide.html (optional, with default settings)). Optimal alignment can be evaluated using any suitable parameters of the selected algorithm, including default parameters.

[0053]

[0058] Complementarity can be complete or moderate / sufficient. Complete complementarity between two nucleic acids may mean that they can form a double helix, where each base in the double helix is ​​bonded to a complementary base by the Watson-Crick pairing rule. Moderate / sufficient complementarity may mean that the sequence of one strand is not entirely and / or completely complementary to the sequence of the other strand, but that sufficient bonding 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 conventional methods.

[0054]

[0059] As used herein, the term "hybridization" generally refers to a reaction in which one or more polynucleotides react to form a complex stabilized by hydrogen bonds between the bases of nucleotide residues. Hydrogen bonds can be formed by Watson-Crick base pairing, Hoogsteen-type bonding, or any other bond based on base complementarity. This can occur in a sequence-specific manner. The complex may consist of two strands forming a double-stranded structure, three or more strands forming a multi-stranded complex, a single self-hybriding strand, or any combination thereof. Hybridization reactions can constitute a step in a broader process, such as the initiation of PCR or the enzymatic cleavage of a polynucleotide by an endonuclease. A second sequence complementary to a first sequence may be referred to as the “complement” of the first sequence. The term “hybridizable,” when applied to polynucleotides, generally refers to the ability of a polynucleotide to form a complex stabilized by hydrogen bonds between the bases of its nucleotide residues in a hybridization reaction.

[0055]

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

[0056]

[0061] As used herein, the term “target site” generally refers to a polynucleotide sequence containing a target polynucleotide (or target nucleotide). The target polynucleotide (or target nucleotide) of a target site may be one or more sequence variants. Examples of one or more sequence variants may include a single nucleotide change, an insertion or deletion of one or more nucleotides (e.g., consecutive or discontinuous nucleotides), copy number variations (CNVs) containing 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, 1kb or less), and microsatellite instability (MSI). The 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. The 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. The 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 compared to the target polynucleotide. The 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 compared to the target polynucleotide. In some examples, the target site may be the target polynucleotide.

[0057]

[0062] As used herein, the term “stringent conditions” generally refers to one or more hybridization conditions under which nucleic acids complementary to the target sequence primarily hybridize with the target sequence and substantially do not hybridize with non-target sequences. Stringent conditions can be sequence-dependent and vary depending on several factors. In some cases, longer sequences may require higher temperatures at which they can specifically hybridize with their target sequences.

[0058]

[0063] As used herein, the term “recognition portion” generally refers to the ability to interact with a “recognition sequence” or “recognition site,” such as a nucleic acid sequence, i.e., a desired (or target) nucleic acid sequence. This refers to molecules (e.g., small molecules, polynucleotides, proteins, variations thereof, or combinations thereof) that possess a recognition moiety. The recognition moiety may include a domain (e.g., a component containing a domain) that has the ability to bind to (e.g., hybridize with) the 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, the recognition moiety may associate with (e.g., bind to) a secondary molecule containing such a domain. In some examples, the recognition moiety may include a nucleic acid molecule that has the ability to hybridize with the recognition sequence. In some examples, the recognition moiety may include, but are not limited to, a component exhibiting specific biological activity, including the activity of one or more nucleases (e.g., double-stranded nucleases), nickases, transcription activators, transcription repressors, nucleic acid methyltransferases, nucleic acid demethyltransferases, and recombinases. 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 nucleotides or more. The recognition sequence may contain at most 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 nucleotides or less.

[0059]

[0064] The recognition portion can be used to isolate a desired molecule containing a desired nucleic acid sequence from multiple molecules (e.g., multiple nucleic acid molecules). The recognition portion can be used to concentrate a desired molecule containing the desired nucleic acid in a composition or reaction mixture. In some examples, the recognition portion can 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 portion may contain biotin, which can form a complex with streptavidin magnetic beads for isolation or concentration.

[0060]

[0065] Examples of recognition moieties include CRISPR-related (Cas) systems (e.g., Cas proteins, including catalytically active or inactive Cas polypeptides); zinc finger nucleases (ZFNs); activator-like effector nucleases (TALENs); meganucleases; RNA-binding proteins (RBPs); Cas RNA-binding proteins; recombinases; flippases; transposases; Argonaut (Ago) proteins (e.g., prokaryotic Argonaut (pAgo), archaeal Argonaut (aAgo), and eukaryotic Argonaut (eAgo)); variants thereof; and combinations thereof. Recognition moieties may include polynucleotides (e.g., sequences of at least 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides or longer) that can be captured by a capture system using one or more interactions, such as 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 may share complementarity with the recognition sequence of the target nucleic acid molecule.

[0061]

[0066] As used herein, the term “nickase” generally refers to a molecule (e.g., an enzyme) that cleaves one strand of a double-stranded nucleic acid molecule (i.e., “inserts a nick” into the double-stranded molecule). A nickase may be a nuclease that cleaves only a single DNA strand, either due to its innate function or because it has been engineered to cleave only a single DNA strand (e.g., modified by mutations and / or deletions of one or more nucleotides). A nickase may be a nicking enzyme (e.g., restriction endonuclease, nicking endonuclease, etc.). A nickase may bind to a nicking site of a double-stranded nucleic acid molecule, creating a nick (or gap) in one strand of that double-stranded nucleic acid molecule. The nick may occur within the nicking site. Alternatively, the nick may occur adjacent to the nicking site. In some cases, a nickase may bind to a nickase binding site adjacent to the nicking site. The nick may be at least 1, 2, 3, 4, 5 in length. 6, 7, 8, 9, 10 nucleotides or more. Nicks can be up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide in length. Examples of nickases include 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 l OI, Nt.Bpu l OI, and Nt,Bst9I, their variations and combinations. Combinations can be listed. In some examples, a nucleic acid molecule (e.g., a double-stranded nucleic acid molecule or a self-complementary single-stranded nucleic acid molecule) may contain at least one nicking site that already contains at least one nick.

[0062]

[0067] The terms “CRISPR-related system,” “Cas system,” and “Cas complex,” as used synonymously herein, generally refer to a two-component ribonucleoprotein complex comprising a guide RNA (gRNA) and a Cas polypeptide or protein (e.g., Cas endonuclease, its catalytic or non-catalytic derivatives, etc.) or another protein having endonuclease activity. The term “CRISPR” refers to clustered, regularly arranged short palindromic sequence repeats and their associated systems. At least a portion of the gRNA may be complementary 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 the target site (or genomic target site). The Cas polypeptide may be guided to the target region by the gRNA pairing (or hybridizing) with the reverse strand (binding site) of the protospacer. The PAM site generally refers to a short sequence recognized by a Cas polypeptide, which may be necessary for nuclease (or nickase) activity. The sequence and number of nucleotides in the PAM site may vary depending on the type of Cas enzyme.

[0063]

[0068] Cas polypeptides may contain nuclease (or nickase) activity, and gRNA may interact with the Cas polypeptide to direct its nuclease (or nickase) activity to a desired target region. Alternatively, Cas polypeptides 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 the CRISPR-related type I, type II, or type III system, or proteins derived therefrom, that may possess RNA-induced polynucleotide binding activity or nuclease activity. Suitable examples of 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, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or Ca Examples include 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, their homologs and modified versions thereof (e.g., catalytic or non-catalytic). Depending on the context, Cas proteins may include Cpf1 (or Cas12a), C2c1 (or Cas12b), C2c2, their homologs, and modified versions thereof (e.g., catalytic or non-catalytic), or proteins derived therefrom from the CRISPR-related type V or type VI system.

[0065]

[0070] The specific examples used herein refer to the Cas protein, but are not endonucleas. Other proteins with ase activity may be used. Such other proteins do not have to be Cas proteins, but may be configured for use with, for example, gRNA.

[0066]

[0071] Cas polypeptides or proteins can be manipulated to change their nuclease activity to nickasase activity. For example, Cas9 derived from Streptococcus pyogenes. Substitution of aspartic acid with alanine in the RuvC I catalytic domain (D10A) can convert Cas9 from a double-strand cleavage nuclease to a single-strand cleavage Cas9n nickase. This Cas9n nickase mutant can introduce single-strand breaks targeted by gRNA into DNA instead of the double-strand breaks produced by the wild-type Cas polypeptide. Other examples of Cas9 nickase mutations include H840A, N854A, and N863A.

[0067]

[0072] As used herein, the term “guide RNA (gRNA)” generally refers to an RNA molecule that can bind to a Cas polypeptide and help target the Cas polypeptide to a specific location within a target nucleic acid region (e.g., DNA or a gene). The degree of complementarity between the gRNA and the specific location within the target nucleic acid region may be at least 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or greater. Guide RNA may include a CRISPR RNA (crRNA) segment and a transactivating crRNA (tracrRNA) segment. As used herein synonymously, the terms “crRNA” and “crRNA segment” generally refer to an RNA molecule or 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 synonymously herein, generally refer to an RNA molecule or portion thereof containing a protein-binding segment (e.g., a protein-binding segment capable of interacting with CRISPR-related proteins such as Cas9). In some cases, the guide RNA may be a single guide RNA (sgRNA), where the crRNA segment and tracrRNA segment are located on the same RNA molecule. The gRNA may contain one or more peptide nucleic acids.

[0068]

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

[0069]

[0074] As used herein, the term "polymerase" generally refers to an enzyme (e.g., natural or synthetic) that has the ability to catalyze polymerization reactions. Examples of polymerases include nucleic acid polymerases (e.g., DNA polymerase or RNA polymerase), transcriptionases, and ligases. A polymerase can be a polymerization enzyme. The term "DNA polymerase" generally refers to an enzyme that has the ability to catalyze the polymerization reaction of DNA.

[0070]

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

[0071]

[0076] The terms “sequence variant” and “sequencing variant,” as used synonymously herein, generally refer to any variation in a sequence compared to one or more reference sequences. Typically, for a given population of individuals from which a reference sequence is provided, sequence variants occur at a lower frequency than that reference sequence. For example, a particular bacterial genus may have a consensus reference sequence for the 16S rRNA gene; however, a particular species within that genus may have one or more sequence variants within the gene or a portion of the gene that are useful in identifying that species within a bacterial population. As a further example, sequences from multiple individuals of the same species or multiple sequencing reads from the same individual may, when optimally aligned, produce a consensus sequence, and sequence variants relating to that consensus may be used to identify mutants in the population that represent dangerous contamination. Generally, a “consensus sequence” refers to a nucleotide sequence that reflects the most common base selection at each position in the sequence when a set of related nucleic acids are subjected to intensive mathematical analysis and / or sequence analysis, such as an optimal sequence alignment following one of various sequence alignment algorithms. A reference sequence can be a single reference sequence, such as a predetermined genome sequence from a single individual. A reference sequence can be a consensus sequence formed by aligning multiple sequences, such as predetermined genome sequences from multiple individuals serving as a reference population, or multiple sequencing reads of polynucleotides from the same individual. A reference sequence can be a consensus sequence formed by optimally aligning sequences from a sample under analysis, such as a sequence variant corresponding to a variation compared to a corresponding sequence in the same sample. Sequence variants can occur at low frequencies in a population (also referred to as "rare" sequence variants). For example, sequence variants can occur at frequencies 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%, or less than 0.001%. Sequence variants can occur at a frequency of less than 0.1%.

[0072]

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

[0073]

[0078] As used herein, the term “sequencing” generally refers to the procedure for determining the order in which nucleotides appear in a target nucleotide sequence. Sequencing methods may 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 may be useful for obtaining millions of sequences in a single run. In some cases, sequencing may be performed using one or more nanopore sequencing methods, such as sequencing bisynthesis, sequencing biligation, or sequencing bicrevage.

[0074]

[0079] As used herein, the term “nanopore” generally refers to a pore, channel, or passage formed in a membrane or otherwise provided. The membrane may be an organic membrane, such as a lipid bilayer, or a synthetic membrane, such as a membrane formed from a polymer material, such as a protein nanopore. The membrane may also be a solid membrane (e.g., a silicon substrate). Nanopores may be located adjacent to or near a sensing circuit or an electrode coupled to a sensing circuit, such as a complementary metal-oxide-semiconductor (CMOS) or field-effect transistor (FET) circuit. Nanopores may be part of a sensing circuit. Nanopores may have an intrinsic width or diameter ranging from, for example, about 0.1 nanometers (nm) to 1000 nm. Nanopores may be biological nanopores, solid nanopores, hybrid biological-solid nanopores, variations thereof, or combinations thereof. Examples of biological nanopores include, but are not limited to, OmpG-like nanopores from Escherichia coli sp., Salmonella sp., Shigella sp., and Pseudomonas sp. Alpha-hemolysin (α-hemolysin) from Staphylococcus aureus sp., smegma MspA from the bacterial species (M. smegmatis sp), their functional variants, or combinations thereof Examples include forward sequencing and / or reverse sequencing. Examples of solid nanopores include, but are not limited to, silicon nitride, silicon oxide, graphene, molybdenum sulfide, functional variants thereof, or combinations thereof. Solid nanopores can be fabricated by high-energy beam fabrication, imprinting (e.g., nanoimprinting), laser ablation, chemical etching, plasma etching (e.g., oxygen plasma etching), etc.

[0075]

[0080] As used herein, the term “nanopore sequencing complex” generally refers to a nanopore linked or coupled to an enzyme, such as a polymerase, which then associates with a polymer, such as a polynucleotide template. The nanopore sequencing complex may be located in a membrane, such as a lipid bilayer, in which case it plays a role in identifying polymer components, such as nucleotides or amino acids.

[0076]

[0081] The terms “nanopore sequencing” and “nanopore-based sequencing,” as used synonymously herein, generally refer to methods for determining the sequence of polynucleotides using nanopores. In some cases, the sequence of polynucleotides 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 apparatus.

[0077]

[0082] As used herein, the term “barcode” generally refers to a given nucleic acid sequence that enables the identification of some feature of the polynucleotide to which the barcode is associated (e.g., a polynucleotide containing at least a portion of the barcode or a polynucleotide complementary to at least a portion of the barcode). In some examples, the feature of the polynucleotide to be 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, or 20 nucleotides long or more. The barcode may be at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleotides long. A barcode associated with a polynucleotide from a first sample may differ from a barcode associated with a polynucleotide from a second sample different from the first sample (e.g., different sequence and / or different length). In that case, identifying the barcode of each polynucleotide may facilitate the identification of one or more sample sources of the polynucleotide. Thus, different samples with different barcodes can be analyzed together (e.g., in batches) (e.g., sequenced) and separated, at least partially, based on the barcodes during the analysis. In some cases, the barcode may be a mutation, insertion or other change in one or more nucleotides in the barcode sequence. It can be accurately identified even after deletions (e.g., mutations, insertions, or deletions of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides or more). 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 3 nucleotide positions, such as at least 3, 4, 5, 6, 7, 8, 9, 10 nucleotide positions or more. Multiple barcodes may appear in a pool of samples, and one or more barcodes contained in a polynucleotide from each sample will differ from barcodes contained in polynucleotides from other samples in the pool. Samples of polynucleotides containing one or more barcodes can be pooled based on the barcode sequence to which they are concatenated, such that all four nucleotide bases A, G, C, and T appear approximately equally at one or more positions along each barcode in the pool (such as at 1, 2, 3, 4, 5, 6, 7, 8 or more positions in the barcode, or all positions). In some examples, the methods of the present disclosure may include identifying the original sample from which the target polynucleotide originates based on a barcode sequence to which the target polynucleotide is ligated. The barcode may include a nucleic acid sequence that, when ligated to the target polynucleotide, can serve to identify the original sample from which the target polynucleotide originates. In some examples, an oligonucleotide primer (e.g., an amplification primer) may include one or more barcodes. In other examples, a nucleic acid molecule is coupled (e.g., ligated) to an adapter nucleic acid (e.g., for cyclization), and the adapter nucleic acid may include one or more barcodes.

[0078]

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

[0079]

[0084] As used herein, the term “subject” generally refers to the original individual or entity from which the sample originates, such as a vertebrate (e.g., a mammal such as a human) or an invertebrate. Mammals may be mice, monkeys, humans, farm animals (e.g., cows, goats, pigs, or chickens) or pets (e.g., cats or dogs). A subject may be a plant. A subject may be a patient. A subject may be asymptomatic with respect to a disease (e.g., cancer). Alternatively, a subject may be symptomatic with respect to a disease.

[0080]

[0085] Whenever the terms "at least," "greater than," or "greater than or equal to" precede the first number in a sequence of two or more numbers, those terms apply to each of the numbers in that sequence. 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 terms “not greater than,” “less than,” or “less than or equal to” precede the first number in a sequence of two or more numbers, those terms apply to each of the numbers in that sequence. For example, 3, 2, or 1 less than or equal to is equal to 3 less than or equal to 3 less than or equal to 2 less than or equal to 1 less than or equal to 1.

[0082]

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

[0088] In one embodiment, 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 indicating the impedance or impedance change of the sensor when at least a portion of the target molecule is bound to or in close proximity to at least a portion of the sensor. One or more signals may be available for analyzing or identifying 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 fewer sensors.

[0084]

[0090] The detected signal, which indicates the impedance or impedance change of a sensor induced by the target molecule, may be a single measurement. Alternatively, the detected signal may be the median or mean of multiple measurements.

[0085]

[0091] When detecting one or more signals indicating the impedance or impedance change of the sensor, at least a portion of the target molecule may bind to the binding site of the sensor. The binding site 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 site. 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 sites. 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 fewer sensors.

[0086]

[0092] Figures 1A to 1E schematically illustrate an example of a system including the sensor of this disclosure for evaluating (e.g., analysis, identification, quantification, ranking, etc.) one or more target molecules. Referring to Figure 1A, the sensor may be immersed in a liquid 105 (e.g., water or buffer, e.g., physiological saline, high-electrolyte buffer, low-electrolyte buffer, etc.). The sensor may include a sensing electrode 110 electrically in 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 liquid 105, which is in the vicinity of or adjacent to the sensing electrode 110. Target molecules (designated 102-1, 120-2, and 120-3) may be present in or suspended in the liquid. The target molecules may or may not be coupled to a probe (e.g., a fluorescent tag or redox species). Referring to Figure 1B, when at least a portion of any of the target molecules (e.g., target molecule 120-1) is adjacent to or enters the volume 112 of liquid 105, one or more signals indicating the impedance or impedance change of volume 112 may be detected by the sensor.

[0087]

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

[0088]

[0094] For example, the detected impedance or impedance change between the sensing electrode 110 and the reference electrode 115 is 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, and 100 microohms. Ohm, 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 0, 40 milliohms, 50 milliohms, 60 milliohms, 70 milliohms, 80 milliohms, 90 milliohms, 100 milliohms, 200 milliohms, 300 milliohms, 400 milliohms, 500 milliohms, 600 milliohms, 700 milliohms, 800 milliohms, 900 milliohms, 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, 1 kilohm, 2 kilohms, 3 kilohms, 4 kilohms, 5 kilohms, 6 kilohms, 7 kilohms, 8 kilohms, 9 kilohms Ohms, 10 kilohms, 20 kilohms, 30 kilohms, 40 kilohms, 50 kilohms, 60 kilohms, 70 kilohms, 80 kilohms, 90 kilohms, 100 kilohms, 200 kilohms, 300 kilohms, 400 kilohms, 500 kilohms, 600 kilohms, 700 kilohms, 800 kilohms, 900 kilohms, 1,000 kilohms, or more.For example, the detected impedance or impedance change between the sensing electrode 110 and the reference electrode 115 can range from approximately 1,000 kΩ, 900 kΩ, 800 kΩ, 700 kΩ, 600 kΩ, 500 kΩ, 400 kΩ, 300 kΩ, 200 kΩ, 100 kΩ, 90 kΩ, 80 kΩ, 70 kΩ, 60 kΩ, 50 kΩ, 40 kΩ, 30 kΩ, 20 kΩ, 10 kΩ, 9 kΩ, 8 kΩ, and 7 kΩ. Milliohms, 6 kilohms, 5 kilohms, 4 kilohms, 3 kilohms, 2 kilohms, 1 kilohm, 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 It 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 is 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 Measurements can be taken over periods of time such as milliseconds, 20 milliseconds, 30 milliseconds, 40 milliseconds, 50 milliseconds, 60 milliseconds, 70 milliseconds, 80 milliseconds, 90 milliseconds, 100 milliseconds, 200 milliseconds, 300 milliseconds, 400 milliseconds, 500 milliseconds, 600 milliseconds, 700 milliseconds, 800 milliseconds, 900 milliseconds, 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, or longer (e.g., a single measurement, multiple measurements for calculating the average of multiple measurements).For example, the detected impedance or impedance change between the sensing electrode 110 and the reference electrode 115 can last up to approximately 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, 200 microseconds, 100 microseconds, 90 microseconds, and 80 microseconds. Measurements may be taken over a period of time of 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, 20 nanoseconds, 10 nanoseconds, 9 nanoseconds, 8 nanoseconds, 7 nanoseconds, 6 nanoseconds, 5 nanoseconds, 4 nanoseconds, 3 nanoseconds, 2 nanoseconds, 1 nanosecond, or less (e.g., a single measurement, multiple measurements for calculating the average of multiple measurements).

[0090]

[0096] Referring to Figures 1A and 1B, an electric field may be applied between the sensing electrode and the reference electrode. Optionally, a function generator may be operably coupled to the sensor, which may apply a voltage (e.g., a small-amplitude sinusoidal voltage), causing a current to be generated in the sensor's circuit. Subsequently, the current, as well as its amplitude and phase (of the alternating current (i.e., AC) component), may be measured. Such values ​​may be converted into one or more signals indicating the impedance or impedance change of the sensor. Measurements may be repeated at a series of different voltages (or frequencies), so that the impedance spectrum of the sensor can be accumulated. Optionally, an LCR meter may be operably coupled to the sensor, and one or more LCR signals or signatures of the sensor may be measured when a target molecule comes into contact with or near the sensing electrode (e.g., directly or indirectly).

[0091]

[0097] Referring to Figure 1C, the sensing electrode 110 of the sensor may be positioned adjacent to a substantially flat and planar surface 130. Referring to Figure 1D, the sensing electrode 110 of the sensor may be positioned within the well 135, for example, at the bottom of the well 135. The well 135 may be a closed-bottom well. Optionally, at least a portion of the well surface (e.g., a portion of the well surface not covered by the sensing electrode) may be covered with a dielectric material (e.g., an insulating material). Alternatively, the well surface may not be covered with a dielectric material. The well may facilitate the localization or capture of the target molecule within the well over a longer period and / or with higher binding affinity, thus improving the accuracy and sensitivity of the sensor. Optionally, (i) a first target molecule may be captured in the well, and a first signal indicating the impedance or impedance change of the sensor may be measured, and (ii) A subsequent target molecule may be trapped in the well, and an additional signal indicating the impedance or impedance change of the sensor may be measured. In (ii), the first target molecule may remain trapped in the well. For example, the target molecule may bind to a substrate placed in the well. Alternatively, the first target molecule may not be trapped in the well in (ii). For example, the first target molecule may leave the well before (ii). Referring to Figure 1E, the sensing electrode 110 of the sensor may be located beneath a nanopore (e.g., a protein nanopore or a solid nanopore). In this example, the sensing electrode 110 may be located beneath a protein nanopore 140 embedded in a lipid bilayer 145. The lipid bilayer 145 may or may not have capacitance. The nanopore 140 is configured to function as a charge and / or size filter, so that it is possible to control what passes through the nanopore 140 into the well 135 and induces a change in the sensor's LCR properties. 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 facilitate the filtering out of positively charged molecules, allowing only negatively charged molecules to enter and interact with the sensing electrode 110. In some cases, the sensor, as shown in Figure 1E, may detect one or more signals indicating impedance or impedance change within the sensor after (i) the target molecule (or tag initially coupled to the target molecule) has passed through the nanopore 140, and (ii) the passed target molecule (or tag) has coupled (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 inside the nanopore 140. In one example, in detecting 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, in detecting one or more signals, at least a portion of the target molecule (or tag) may be in close proximity to a conductive material.

[0092]

[0098] In another embodiment, the Disclosure provides a system for analyzing or identifying a target molecule. The system may include a sensor comprising a sensing electrode and a reference electrode electrically connected to each other. The sensor may include a dielectric material coupled to the sensing electrode and covering a first portion of the surface of the sensing electrode. The sensor may include a conductive material coupled to the sensing electrode and covering a second portion of the surface of the sensing electrode. The sensor may include a binding unit coupled to the conductive material, the binding unit configured to bind to a target molecule. The sensor may be configured to detect one or more signals indicating the impedance of the sensor or a change in impedance when at least a portion of the target molecule is bound to the binding unit. One or more signals may be available for analyzing or identifying the target molecule. The conductive material may be a bond (e.g., a chemical bond) or may include a binding unit (e.g., a nanorod, peptide, small molecule, etc.) of any desired dimensions (e.g., length, cross-sectional diameter or area, volume, etc.). In an alternative embodiment, the binding unit may be directly coupled to the sensing electrode. In yet another embodiment, the bonding unit may be coupled to at least a portion of the dielectric material that is coupled to the sensing electrode.

[0093]

[0099] One or more signals may indicate (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. One or more signals may indicate at least two 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. One or more signals may indicate (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] One or more signals may be current or voltage. One or more signals may be current and voltage. It can be a pressure. One or more signals do not have to be tunnel currents.

[0095]

[0101] The first portion of the sensing electrode coated with dielectric material may be at least 50 percent (%) of the surface of the sensing electrode. In some cases, the first portion of the sensing electrode may 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 may be at most 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. Depending on the circumstances, 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. Depending on the circumstances, 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 dimensions of the sensing electrode (or the surface area coupled to the dielectric and conductive materials) may be no more than 100 times the average size of the target molecule. In some cases, the average cross-sectional dimensions of the sensing electrode may 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 time, 0.5 times, or 0.1 times the average size of the target molecule. In some cases, the average cross-sectional dimensions of the sensing electrode may be at least 0.1 times, 0.5 times, 1 time, 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 molecule. Depending on the circumstances, the average cross-sectional dimensions of the sensing electrode may 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 greater than the average size of the target molecule. Depending on the circumstances, the average cross-sectional dimensions of the sensing electrode may 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 than the average size of the target molecule.

[0098]

[0104] Alternatively, the average cross-sectional dimensions of the sensing electrode can be smaller than the average size of the target molecule. In some cases, the average cross-sectional dimensions 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 molecule. In some cases, the average cross-sectional dimensions 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 molecule.

[0099]

[0105] The area of ​​the second portion of the sensing electrode surface is 500 square angstroms (Å). 2 ), 100Å2 , 50 Å 2 , 10 Å 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 Or it can be less than or equal to the following. In some cases, 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 the atoms of the conductive material (e.g., twice the van der Waals radius).

[0100]

[0106] The dielectric material can be a solid layer (e.g., a solid metal or semiconductor material) or a self-assembled monolayer film (SAM).

[0101]

[0107] The conductive material can be a single molecule (e.g., a single conductive polymer chain). In some cases, one or more features of an atomic force microscope (AFM) (e.g., the piezoelectric cantilever probe of the AFM) for coupling a single molecule to a specific (or random) position within the surface of the sensing electrode. Instead, 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 a maximum of 100,000, 50,000, 10,000, 5,000, 1,000, 500, 100, 50, 10, 5 or 1 molecule.

[0102]

[0108] Depending on the circumstances, a substrate may bind to the binding unit of the sensor of the Disclosure (e.g., a nuclease or a variant thereof), and the binding unit or additional elements (e.g., additional enzymes) may be configured to cleave at least a portion of the substrate. The sensor may be configured to (i) detect the difference of one or more signals indicating a change in the impedance 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, antibody, aptamer, non-biological material (e.g., synthetic polymer), functional fragments thereof, functional variants thereof, or a combination thereof. The enzyme may be selected from the group consisting of polymerases, nucleases (e.g., double-chain nucleases), nicasses, transcription activators, transcription repressors, nucleomethylases, nucleomethylases, and recombinases. The antibody may be a whole antibody or its antigen-binding fragment, e.g., scFv, Fab fragment, VHH domain, or VH domain of a heavy chain monoantibody. The antibody may be monospecific or multispecific (e.g., bispecific, trispecific, etc.). The antibody may be monovalent or polyvalent (e.g., bivalent, trivalent, etc.).

[0104]

[0110] The bonding unit may be directly coupled to a conductive material, for example, by covalent or non-covalent bonding to the conductive material. Alternatively, the bonding unit may be indirectly coupled to the sensing electrode via a linker, for example, which bonds to the conductive material on one side and to the bonding unit on the other. In such a case, the linker may also be conductive to minimize interference with the sensor's sensing capability. Alternatively, the linker may not be conductive.

[0105]

[0111] The target molecule may be selected from the group consisting of small molecules, nucleotides, polynucleotides, amino acids, peptides, polypeptides, their variants, and combinations thereof.

[0106]

[0112] A target molecule may contain one or more tags. A target molecule may contain at least one, two, three, four, five, or more tags. A target molecule may contain up to five, four, three, two, or one tags. A tag 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 target molecule having each tag may be substantially distinguishable from others. In some cases, one or more features or properties of a target molecule (e.g., size, shape, charge, vibration, motion in a fluid) may further influence one or more signals of the sensor, thereby making one or more signals of each target molecule having each tag more easily distinguishable from others. In some cases, the tags of this disclosure may be impedance tags. An impedance tag may be configured to produce a change in the impedance detected in 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 We can list the following combinations.

[0107]

[0113] Alternatively, the target molecule may not contain any tags. In such cases, one or more features or properties of the target molecule (e.g., size, shape, charge, vibration, motion in a fluid) may further influence one or more signals of the sensor, thereby making each of the one or more signals of target molecules with their respective tags more easily distinguishable from others. In one 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 that binds to a cyclic nucleic acid for rolling circle amplification (RCA) (e.g., a polymerase configured to perform RCA). The target portion may be a nucleic acid base without any fluorescent or redox species tags; however, the sensitivity of the sensor of this disclosure may have the ability to analyze one or more signals indicating the impedance or impedance change of the sensor when a nucleotide is bound to a cyclic nucleic acid by a polymerase.

[0108]

[0114] The size, shape, and / or resolution of one or more components of the sensor disclosed herein may be defined or limited by the resolution of the technique, such as the size of photolithography, etching, nanoimprinting, or bionanopores.

[0109]

[0115] Figures 2A to 2E schematically illustrate an example of a system including the sensor of the present disclosure for, for example, evaluating (e.g., analysis, identification, quantification, ranking, etc.) one or more target molecules. Referring to Figure 2A, the sensor may be immersed in a liquid 105 (e.g., water or buffer, e.g., saline, high-electrolyte buffer, low-electrolyte buffer, etc.). The sensor may include a sensing electrode 110 that is electrically in communication with a reference electrode 115. The sensor may include a dielectric material (e.g., SAM) 205 coupled to at least the upper surface of the sensing electrode 110. The SAM 205 may cover most of the upper 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 upper surface of the sensing electrode 110. The conductive material 210 may be embedded in the "sea" of SAM 205. The conductive material 210 may also have a coupling portion 215 coupled to it on the opposite side (or end) from the sensing electrode 110. The sensor may be configured to detect or measure one or more LCR properties or changes thereof within a volume 217 of liquid 105, where this volume 217 is in the vicinity of or adjacent to a binding unit 215. In this example, the binding unit 215 may be an enzyme (e.g., 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., SAM205 as shown in Figure 2A) coupled to at least a portion of the surface of the sensing electrode is at least approximately 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, 500nm, 600nm, 700nm, 800nm, 900nm, 1μm, 2μm, 3μm, 4μm, 5μm, 6μm, 7μm, 8μm, 9μm, 10μm, 20μm, 30μm, 40μm, 50μm, 60μm, 70μm, 80μm, 90μm, 100μm, 200μm, 300μm, 400μm, 500μm, 600μm, 700μm, 800μm, 900μm, 1,000μm or more. The thickness of the dielectric material coupled to at least a portion of the surface of the sensing electrode is approximately 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. The wavelength may be 30nm, 20nm, 10nm, 9nm, 8nm, 7nm, 6nm, 5nm, 4nm, 3nm, 2nm, 1nm, 0.5nm, 0.1nm, or less.

[0111]

[0117] Referring to Figure 2B, the target molecule 230 may be three nucleotide probes (labeled 1, 2, and 3) suspended in liquid 105. Referring to Figure 2C, a binding unit 215 (e.g., polymerase) coupled to the sensing electrode 110 and embedded in a SAM (not shown) may interact with the target nucleotide probe 230-1, thereby coupling the target nucleotide probe to the nucleotide substrate 220. The sensor may measure one or more signals indicating 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 the bulk impedance or impedance change. In some examples, the detected impedance or impedance change is not in the Debye layer (or bilayer) of the electrode, e.g., the sensor electrode 110 as shown in Figure 2C. In some cases, the detected impedance or impedance change may be outside the Debye layer of the electrode. In some cases, the detected impedance or impedance change is not in the Debye layer (or bilayer) of the dielectric material coupled to the sensing electrode (e.g., SAM205 as shown in Figure 2A). In some cases, the detected impedance or impedance change may be outside the Debye layer of the dielectric material.

[0112]

[0118] Referring to Figure 2D, the target molecule 230 may be four nucleotide probes (labeled 240-1, 240-2, 240-3, and 240-4) suspended in liquid 105. Here, the nucleotide probes have the same nucleotide base (e.g., adenine, i.e., A) but different tags, as depicted by tags of different shapes. At a given position (i.e., t=N) along the length of the nucleotide substrate 220, a binding unit 215 (e.g., polymerase) coupled to the sensing electrode 110 and embedded in SAM (not shown) may interact with the target nucleotide probe 240-4 (e.g., by random selection based on the free motion of the nucleotide probes in liquid 105) to couple the target nucleotide probe 240-4 to the nucleotide substrate 220. Subsequently, the sensor may detect one or more signals (e.g., capacitance vs. resistance plot) indicating the impedance or impedance change of the sensor resulting from 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), if the substrate again recruits the same nucleic acid base as before, the binding unit 215 may interact with another target nucleotide probe 240-1 (e.g., by random selection) to couple the target nucleotide probe 240-1 to the nucleotide substrate 220. Subsequently, the sensor may detect one or more signals (e.g., capacitance-resistance plots) indicating the impedance or impedance change of the sensor resulting from the addition of the nucleotide probe 240-1 to the substrate 220.

[0113]

[0119] Depending on the circumstances, the sensor may be configured to detect one or more signals indicating 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 coupled (for example, directly or indirectly via the bonding unit 215 and the conductive material 210) to at least a portion of the sensor, for example, the sensing electrode 110. Alternatively, the sensor may be configured to detect one or more signals indicating 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 coupled to at least a portion of the sensor, for example, the sensing electrode 110, but is in close proximity to it. - It can be configured to detect one or more signals indicating a change in dance.

[0114]

[0120] The sensor of this disclosure has a distance between (i) at least a portion of a target molecule (and / or a tag coupled to the target molecule) and (ii) a sensing electrode of 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 nm When the impedance is 0nm, 1μm, 2μm, 3μm, 4μm, 5μm, 6μm, 7μm, 8μm, 9μm, 10μm, 20μm, 30μm, 40μm, 50μm, 60μm, 70μm, 80μm, 90μm, 100μm, 200μm, 300μm, 400μm, 500μm, 600μm, 700μm, 800μm, 900μm, 1,000μm or greater, the system can be configured to detect even more signals indicating impedance or impedance changes between the sensing electrode and the reference electrode. The sensor as disclosed herein has a maximum distance of approximately 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 When the impedance becomes μm, 900nm, 800nm, 700nm, 600nm, 500nm, 400nm, 300nm, 200nm, 100nm, 90nm, 80nm, 70nm, 60nm, 50nm, 40nm, 30nm, 20nm, 10nm, 9nm, 8nm, 7nm, 6nm, 5nm, 4nm, 3nm, 2nm, 1nm, 0.5nm, 0.1nm or less, the system can be configured to detect even more signals indicating impedance or impedance changes between the sensing electrode and the reference electrode.

[0115]

[0121] The sensors of this disclosure may be configured to detect more signals indicating impedance or impedance changes between the 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 Figure 2A) in the vicinity of or adjacent to the sensing electrode. The predetermined space is at least about 0.1 nm 2 , 0.5nm 2 , 1nm, 2nm 2 , 3nm 2 , 4nm 2 , 5nm 2 , 6nm 2 , 7nm 2 , 8nm 2 , 9nm 2 , 10nm 2 , 20nm 2 , 30nm 2 , 40nm 2 , 50nm 2 , 60nm 2 , 70nm 2 , 80nm 2 , 90nm 2 , 100nm 2 , 200nm 2 , 300nm 2 , 400nm 2 , 500nm 2 , 600nm 2 , 700nm 2 , 800nm 2 , 900nm 2 , 1 μm 2 , 2μm 2 , 3μm 2 , 4μm 2 , 5μm 2 , 6μm 2 , 7μm 2 , 8μm 2 , 9μm 2 , 10 μm 2 , 20 μm 2 , 30 μm 2 , 40 μm 2 , 50 μm 2 , 60 μm 2 , 70 μm 2 , 80 μm 2 90 μm2 、100 μm 2 、200 μm 2 、300 μm 2 、400 μm 2 、500 μm 2 、600 μm 2 、700 μm 2 、800 μm 2 、900 μm 2 、1,000 μm 2 or may be characterized by having a volume greater than that. The predetermined space is up to about 1,000 μm 2 、900 μm 2 、800 μm 2 、700 μm 2 、600 μm 2 、500 μm 2 、400 μm 2 、300 μm 2 、200 μm 2 、100 μm 2 、90 μm 2 、80 μm 2 、70 μm 2 、60 μm 2 、50 μm 2 、40 μm 2 、30 μm 2 、20 μm 2 、10 μm 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 、900 nm 2 、800 nm 2 、700 nm 2 、600 nm 2 、500 nm 2 、400 nm 2 、300 nm 2 、200 nm 2 、100 nm 2 、90 nm 2 、80 nm 2 、70 nm 2 、60 nm 2 、50 nm2 , 40nm 2 , 30nm 2 , 20nm 2 , 10nm 2 , 9nm 2 , 8nm 2 , 7nm 2 , 6nm 2 , 5nm 2 , 4nm 2 , 3nm 2 , 2nm 2 , 1nm 2 , 0.5nm 2 , 0.1nm 2 Or it may be characterized by having a volume less than that.

[0116]

[0122] The sensors of this disclosure may not require at least a portion of the 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 nanopore) in order to detect one or more signals indicating impedance or impedance change. For example, as shown for example in Figures 2A to 2D, the sensor may not contain a nanopore or be operably coupled to one. Instead, at least a portion of the target molecule (and / or a tag coupled to the target molecule) may enter and / or pass through the sensor's pore (e.g., a nanopore such as a protein nanopore or a solid nanopore) in order for the sensor to detect one or more signals indicating impedance or impedance change. For example, as shown for example in Figure 1E, the sensor may contain a nanopore.

[0117]

[0123] Figure 2E shows an example of capacitance-to-resistance plots obtained by the sensor after each of the nucleotide probes 240-4 and 240-1 has been added to the substrate 220 by the binding unit 215. Measurements of the impedance signal of liquid 105 may be shown as background or control. As shown by the plot in Figure 2E, although coupled to the same nucleotide base (e.g., adenine), different tags result in different capacitance-to-resistance characteristics, thereby enabling the sensor to decompose the sequential addition of two or more identical nucleic acid bases to the substrate 220. In one example, the sensor of the present disclosure may be able to decompose the difference between (i) a first time (t) showing the addition of the Nth adenine (e.g., the 20th adenine) and (ii) a second time showing the addition of the subsequent (N+1)th adenine (e.g., the 21st adenine).

[0118]

[0124] In some embodiments, as shown in Figure 3, the height 310 of the channel 305 holding the fluid 105 may be designed to suppress the mobility (or diffusion) of target molecules along an axis parallel to the height 310 of the channel 305. Limiting the effective operating area of ​​the sensor (e.g., the dimensions of the binding unit, conductive material, or sensing electrode) can cause any mobility of target molecules along an axis perpendicular to the effective operating area of ​​the sensor (e.g., an axis parallel to the height 310) to negatively affect the measurement of one or more signals. Therefore, by reducing the height 310 of the channel 305, the system can be made substantially two-dimensional, thereby limiting any effect of vertical motion of target molecules relative to the sensing electrode 110. The channel height 310 may 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 may 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 greater.

[0119]

[0125] With respect to Figure 3, the height 310 of channel 305 may, in some cases, be a predetermined height (e.g., during the manufacturing of the sensor). Alternatively, the height 310 of channel 305 may be adjustable. In some cases, it may be desirable to adjust the height 310 of channel 305 according to the size of the target molecule or the average size of the target molecule population. In such cases, one or more switches (e.g., mechanical switches, microfluidic switches) may be placed on or adjacent to at least a portion of the sensor so that the height 310 can be changed in real time. Examples of switches include, but are not limited to, thermal or piezoelectric actuators, electric actuators, etc.

[0120]

[0126] In some embodiments, the sensing electrode and the reference electrode are aligned along a first direction. This may provide a first electric field. In addition, the system may further include an additional 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 from the first direction of the first electric field (e.g., substantially perpendicular). The difference between the first and second directions may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170 degrees or more. The difference between the first and second directions can be up to approximately 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 degree, or less. In some examples, the difference between the first and second directions can be approximately 90 degrees (i.e., the first and second directions can be substantially orthogonal to each other). Figure 4A schematically illustrates a system including a sensor for evaluating (e.g., analysis, identification, quantification, ranking, etc.) one or more target molecules, where the sensor does not include any additional field generators. 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 binding units (e.g., polymerase enzymes) may not be able to contact one or more positions on the substrate 220, preventing the addition of target nucleotides, which can negatively affect the sensitivity and / or accuracy of the sensor. In contrast, Figure 4B schematically illustrates a different system for evaluating one or more target molecules (e.g., analysis, identification, quantification, ranking, etc.), which includes a sensor and an additional field generator configured to apply an additional electric field 405 between an additional pair of electrodes 410 and 415. 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 can unfold and / or stretch the substrate 220 along the direction of the electric field 405, thereby reducing the folding and overlapping of the substrate 220 by the surfaces of the bonding unit 215, conductive material (not shown), and / or sensing electrode 110.

[0121]

[0127] This system may include at least one, two, three, four, five, or more additional field generators. The system may include up to five, four, three, two, or one additional field generator. When including multiple additional field generators, these additional field generators may apply multiple electric fields along the same or different directions. Alternatively, the system may not include any additional field generators.

[0122]

[0128] In some embodiments, the target molecule may contain one or more redox moieties (e.g., as tags), and the sensor of this disclosure may be configured to measure the redox potential (e.g., reduction potential or oxidation potential) of the redox moieties. In some examples, the sensor may be configured to measure the oxidation potential of an oxidative functional group of the target molecule. Since buffer solutions can negatively affect the activity of binding units (e.g., the enzymatic activity of binding units), it may be desirable to reduce the salt concentration in the solution for some binding units. 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 made of one or more redox moieties to measure the redox potential of a target molecule containing such tags, and accuracy may be improved.

[0123]

[0129] Figure 5A schematically illustrates an example of a system including a sensor that measures the oxidation potential of one or more target molecules for evaluating (e.g., analysis, identification, quantification, ranking, etc.) one or more target molecules. This sensor may include one or more components of the system illustrated in Figure 2A, such as a sensing electrode 110, a reference electrode 115, a liquid 105 (e.g., a salt solution), a binding unit 215 (e.g., polymerase), and a polynucleotide substrate 220. Here, the target molecule to be attached to the substrate 220 by the binding unit 215 is 510 These are synthetic nucleotides A, T, C, and G, as indicated by -1, 510-2, 510-3, and 510-4. The redox moiety of the target molecule may 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, and therefore, when each target molecule containing its respective redox tag is attached to the substrate 220 by the binding unit 215, applying different voltages (e.g., different by a few millivolts) may allow for the determination of which target molecule was attached to the substrate 220 by detecting (i) electron transfer between the redox moiety of the target molecule and (ii) the sensing electrode when, for example, the target molecule bound to the redox moiety is bound to the binding unit and attached to the substrate 220.

[0124]

[0130] Figure 5B schematically illustrates time (t, x axis) versus current (I, y axis) and current (indicated by square peaks) plots at various voltages (indicated as V0, V1, V2, V3, and V4 along the y axis) obtained from the sensor in Figure 5A. In Figure 5B, by superimposing multiple time-versus-current plots at various voltages, the different current signatures produced by different target molecule-redox tag complexes as provided herein are shown. Since the redox tags of the four target molecules 510-1, 510-2, 510-3, and 510-4 can respond to different voltages, the sequence 520 of the desired region of the target substrate 220 can be determined using the respective current signatures thus measured.

[0125]

[0131] In some embodiments, the sensor may be configured to determine the residence time of a target molecule on a binding unit. Depending on the circumstances, different target molecules or different tags coupled to target molecules may exhibit different residence times on the sensor's binding unit, and these residence times may be unique or additional signatures for analyzing or identifying the target molecule.

[0126]

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

[0127]

[0133] One or more signals may be currents or voltages measured from a sensing circuit. One or more signals may be currents and voltages measured from a sensing circuit. A signal may be a tunnel current. Alternatively, a signal may not be a tunnel current. A current may be a Faraday current. Alternatively, a current may not be a Faraday current. A current may be at least 1 picoampere (pA), 10 pA, 100 pA, 1 nanoampere (nA), 10 nA, 100 nA, 1 microampere (mA), 10 mA, 100 mA, or more. A current may be at most 100 mA, 10 mA, 1 mA, 100 nA, 10 nA, 1 nA, 100 pA, 10 pA, 1 pA, or less. Current may range from at least picoamperes (pA), tens of pA, hundreds of pA, nanoamperes (nA), tens of nA, hundreds of nA, microamperes (mA), tens of mA, or more. Current may range from at most tens of mA, mA, hundreds of nA, tens of nA, nA, hundreds of pA, tens of pA, pA, or less. Voltage may range from at least 0.1 millivolts (mV), 0.5 mV, 1 mV, 5 mV, 10 mV, 50 mV, 100 mV, 500 mV, or more. Voltage may range from at most 500 mV, 100 mV, 50 mV, 10 mV, 5 mV, 1 mV, 0.5 mV, 0.1 mV, or less. Voltage may range from at least millivolts (mV), tens of mV, hundreds of mV, or more. The voltage can range from several hundred mV to several tens of mV, or even less than a few hundred mV.

[0128]

[0134] In some embodiments, the sensor of the Disclosure is located on a chip or biochip. An array may be provided as such, such as an existing array. An array of sensors may have any preferred number of any sensors of this disclosure. An 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, or more sensors.

[0129]

[0135] In another embodiment, the Disclosure provides a method for analyzing or identifying a target molecule. The method may include using a sensor to detect one or more signals indicating the 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 using one or more signals to analyze or identify the target molecule. The method may utilize any of the subject systems of the Disclosure as illustrated in Figures 1 to 5.

[0130]

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

[0131]

[0137] Figure 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 indicating the 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 one or more signals.

[0132]

[0138] One or more signals may indicate (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. One or more signals may indicate at least two 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, or (ii) the electrical inductance of the sensor or a change therein. One or more signals may indicate (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] One or more signals may be currents or voltages. One or more signals may be currents and voltages. One or more signals do not have to be tunnel currents.

[0134]

[0140] In some embodiments, a sensing electrode and a reference electrode may provide a first electric field. In addition, the method may further include providing an additional electric field generator. The method uses 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. This may further include applying an electric field.

[0135]

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

[0136]

[0142] II. Samples

[0143] The sample for analysis may contain multiple polynucleotides. Polynucleotides may be single-stranded DNA, double-stranded DNA, or a combination thereof. Polynucleotides may also include genomic DNA, genomic cDNA, cell-free DNA, cell-free cDNA, or any combination thereof.

[0137]

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

[0138]

[0145] Multiple polynucleotides can be single-stranded or double-stranded.

[0139]

[0146] Polynucleotide samples can be derived from any suitable source. For example, samples can be obtained from patients, animals, plants, or the environment, such as naturally occurring or artificial air, water systems, soil, airborne pathogen collection systems, subsurface sediments, groundwater, or sewage treatment plants.

[0140]

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

[0141]

[0148] Polynucleotides 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 polynucleotides in the sample can be less than 50 ng, 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 polynucleotides in the sample can be greater than 0.1 ng, 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 polynucleotides can be, for example, from 0.1 ng to 100 ng, from 1 ng to 75 ng, from 5 ng to 50 ng, or from 10 ng to 20 ng.

[0143]

[0150] The polynucleotides in the sample may be single-stranded, either in their as-obtained state or after processing (e.g., denaturation). Further examples of suitable polynucleotides are described herein, including in relation to any of the various aspects of this disclosure. The sample can be subjected to subsequent steps (e.g., cyclization and amplification) without an extraction step and / or a purification step. For example, a fluid sample can be subjected to a treatment that removes cells without an extraction step to produce a purified liquid sample and a cell sample, from which polynucleotides can be isolated. Various procedures are available for the isolation of polynucleotides, such as by precipitation or nonspecific binding to a substrate, followed by the release of bound polynucleotides by washing the substrate. When polynucleotides are isolated from a sample without a cell extraction step, the polynucleotides will generally be extracellular or "cell-free" polynucleotides that can correspond to dead or damaged cells. Such cellular identity can be used to characterize the original cells or cell populations from which they originate, 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, plants, animals, fungi, protists, Monera, viruses, mitochondria, and chloroplasts. The sample polynucleotide may be isolated from the subject, including cell samples, tissue samples, body fluid samples, or organ samples, or cell cultures derived from any of these, such as cultured cell lines, biopsies, blood samples, cheek swabs, or fluid samples containing cells such as saliva. The subject may be an animal, such as a mammal, including a cow, pig, mouse, rat, chicken, cat, dog, or human. The sample may contain tumor cells, for example, in a sample of tumor tissue from the subject.

[0145]

[0152] The sample does not need to contain intact cells, and can be subjected to a process to remove cells, or polynucleotides can be isolated without a cell extraction step, such as by isolating cell-free polynucleotides like cell-free DNA.

[0146]

[0153] Other examples of sample sources include blood, urine, feces, nostrils, 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 can be independently subjected to the methods of this disclosure, such as duplicate, triplicate, quadruplicate, or more analyses. If the sample is from a subject, the reference sequence may also be derived from the subject, such as a consensus sequence from the sample under analysis or a polynucleotide sequence from another sample or tissue of the same subject. For example, a blood sample may be analyzed for ctDNA mutations, and the reference sequence can be determined by analyzing cellular DNA from another sample from the subject, such as a buccal or skin sample.

[0148]

[0155] Polynucleotides can be extracted from a sample by any suitable method, with or without extraction from cells in the sample.

[0149]

[0156] Multiple 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 appears to be a common finding across various malignancies, not limited to any specific type of cancer. Plasma concentrations of free circulating DNA may be lower in controls compared to patients with or suspected of having the condition. In some cases, plasma concentrations of free circulating DNA may range from 14 ng / mL to 18 ng / mL in controls and from 18 ng / mL to 318 ng / mL in patients with neoplasms.

[0150]

[0157] Apoptotic cell death and necrotizing cell death may be contributing factors to cell-free circulating DNA in body fluids. For example, a significant increase in circulating DNA levels can be observed in the plasma of patients with prostate cancer, as well as in patients with other prostate diseases such as benign prostatic hyperplasia and prostatitis. In addition, circulating tumor D NA can be present in fluids derived from the organ in which the primary tumor develops. In some cases, breast cancer can be detected in mammary duct lavage fluid; colorectal cancer in stool; lung cancer in sputum; and prostate cancer in urine or ejaculate. Cell-free DNA can be obtained from various sources. An exemplary source may be the blood sample of the subject. However, cfDNA or other fragmented DNA can originate from various other sources, including, for example, urine and stool samples, and can be a source of cfDNA, including ctDNA.

[0151]

[0158] In some embodiments, the target molecule may represent a health condition or disease. In some cases, the disease may be a tumor or cancer. Non-limiting examples of antigens that can be bound by the binding units of the subject system 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, adipophyllin, adrenoceptor β3 (ADRB3), AGS-22M6, α-folate receptor, α-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), and 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 / testicular antigen 1 (NY-ESO-1), cancer / testicular 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 (basidine), 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 Pumping 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) Escherichia coli) Shiga toxin type 2, ecto-ADP-ribosyltransferase 4 (ART4), EGF-like module-containing mucin-like hormone receptor 2 (EMR2), EGF-like domain multiple 7 (EGFL7), elongation factor 2 mutant (ELF2M), endotoxin, ephrin A2, ephrin B2, ephrin A receptor 2, epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithelial cell adhesion molecule (EpCAM), epidermal glycoprotein 2 (EGP-2), epidermal glycoprotein 40 (EGP-40), ERBB2, ERBB3, ERBB4, ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), Escherichia coli, ETS translocation variant gene 6 (ETV6-AML) located on chromosome 12p, respiratory syncytial virus F protein, FAP, Ig Fc fragment of A receptor (FCAR or CD89), Fc receptor-like 5 (FCRL5), fetal acetylcholine receptor, fibrin II β chain, fibroblast-activating protein α (FAP), fibronectin extradomain-B, FGF-5, Fms-like tyrosine kinase 3 (FLT3), folate-binding protein (FBP), folate hydrolase, folate receptor 1, folate receptor α, folate receptor β, 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 α chain, GPNMB, GnT-V, growth differentiation factor 8, GUCY2C, heat shock protein 70-2 mutant (mut hsp70-2), hemagglutinin, hepatitis A virus cell receptor 1 (HAVCR1), hepatitis B surface antigen, hepatitis B virus, HER1, HER2 / neu, HER3, hexasaccharide portion 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 scattering 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 λ-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-γ-inducible protein, interleukin-11 receptor α (IL-11Rα), interleukin-13 receptor subunit α-2 (IL-13Ra2 or CD213A2), intestinal carboxylesterase, kinase domain region (KDR), KIR2D, KIT (CD117), L1 cell adhesion molecule (L1-CAM), regmine, leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Lewis Y receptor Original, LFA-1 (CD11a), LINGO-1, lipoteichoic acid, LOXL2, L-selectin (CD62L), lymphocyte antigen 6 complex, gene 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 inhibitor (MIF or MMIF), M-CSF, mammary gland differentiation antigen (NY-BR-1), MCP-1, melanoma carcinoma testis antigen-1 (MAD-CT-1) Melanoma carcinoma testicular antigen-2 (MAD-CT-2), melanoma apoptosis inhibitor (ML-IAP), melanoma-associated antigen 1 (MAGE-A1), mesothelin, mucin 1, cell surface-related (MUC1), MUC-2, mucin CanAg, myelin-associated glycoprotein, myostatin, N-acetylglucosaminyltransferase V (NA17), NCA-90 (granulocyte antigen), nerve growth factor (NGF), neuronal apoptosis-modulating proteinase 1, nerve cell adhesion molecule (NCAM), neurite outgrowth inhibitors (examples) For example, NOGO-A, NOGO-B, 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 (bcr-abl) consisting of a cleavage cluster region (BCR) and Abelson mouse leukemia virus oncogene homolog 1 (Abl), Oryctolagus cuniculus, OX-, 40, oxLDL, p53 mutant, paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), panexin 3 (PANX3), phosphate-sodium cotransporter, phosphatidylserine, placenta-specific 1 (PLAC1) ), platelet-derived growth factor receptor α (PDGF-Rα), platelet-derived growth factor receptor β (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 1 recognized by T cells (MelanA or MART1), P15, P53, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostatic acid phosphatase (PAP), prostate cancer cells, prostain, protease serine 21 (testisin or PRSS21), proteasome (prosome, macropain) subunit, type β, 9 (LMP2) Pseudomonas aeruginosa, rabies virus glycoprotein, RAGE, Ras homolog family member C (RhoC), nuclear factor κ-B ligand receptor activator (RANKL), late glycation end product receptor (RAGE-1), receptor tyrosine kinase-like orphan receptor 1 (ROR1), renal ubiquitous receptor 1 (RU1), renal ubiquitous receptor 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-section 2 (SSX2), T cell receptor, TCRγ alternative leading 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 signaling 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 factor (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 myelocytosis viral oncogene neuroblastoma-derived homolog (MYCN), von Wy. Examples include Rebrandt factor (VWF), Wilms oncoprotein (WT1), the X antigen family, member 1A (XAGE1), β-amyloid, and κ light chain.

[0152]

[0159] III. Nanopore

[0160] The sequencing system may include a reaction chamber containing one or more nanopore devices. The nanopore devices may be individually addressable. Individually addressable nanopores may be individually readable. Individually addressable nanopores may be individually writable. Individually addressable nanopores may be individually readable and individually writable. The system may include one or more computer processors to facilitate sample preparation and various operations of this disclosure, such as polynucleotide sequencing. The processors may be coupled to the nanopore devices.

[0153]

[0161] A nanopore device may include multiple individually addressable sensing electrodes. Each sensing electrode may include a film adjacent to the electrode and one or more nanopores within the film. The nanopores may be part of an integrated circuit, positioned adjacent to electrodes coupled to it, or in a film such as a lipid bilayer positioned close to it for sensing. The nanopores may be associated with individual electrodes and sensing integrated circuits or with multiple electrodes and sensing integrated circuits. The nanopores may include solid nanopores.

[0154]

[0162] The apparatus and systems used in the methods provided herein can accurately detect individual nucleotide incorporation events, such as when nucleotides are incorporated into a growing chain complementary to a template. Nucleotides can be incorporated into a growing polynucleotide chain by enzymes such as DNA polymerase, RNA polymerase, or ligase. Enzymes such as polymerases can result in a polynucleotide chain.

[0155]

[0163] The nucleotide to be added may be complementary to the corresponding template polynucleotide chain that hybridizes to the growing chain. The nucleotide may include, but is not limited to, a tag or tag species coupled at any position on the nucleotide, including phosphate, sugar, or nitrogen-containing base moieties such as γ-phosphate of the nucleotide. In some cases, the tag may be detected while the tag is associated with the polymerase during nucleotide tag incorporation. The tag may continue to be detected after nucleotide incorporation until the tag moves through the nanopore, followed by cleavage and / or release of the tag. The nucleotide incorporation event may release the tag from the nucleotide and pass through the nanopore, where it may be detected. The tag may be released by the polymerase, or may be cleaved / released in any preferred manner, including, but not limited to, cleavage by an enzyme located near the polymerase. In this way, a unique tag is released from each type of nucleotide (i.e., adenine, cytosine, guanine, thymine, or uracil), so that the incorporated base (i.e., A, C, G, T, or U) can be identified. In non-release nucleotide uptake events, tags coupled to the uptaken nucleotides are detected using nanopores. In some cases, the tags move through or near the nanopores, which can then be detected using nanopores.

[0156]

[0164] The methods and systems of this disclosure may enable the detection of polynucleotide incorporation events 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 within a given time. For example, individual polynucleotide incorporation events can be detected using a nanopore device, where each event is associated with an individual nucleic acid base. In another example, a nanopore device can be used to detect events associated with multiple bases. For example, the signal detected 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 in the opposite direction to the direction in which it entered the nanopore. The sequencing device can be configured to actively expel the tag from the nanopore.

[0158]

[0166] In certain sequencing methods, the tag is not released even when a nucleotide incorporation event occurs. A nucleotide incorporation event can present the tag to a nanopore without releasing it. The tag can be detected by the nanopore without being released. The tag can be attached to a nucleotide by a linker of sufficient length to present the tag to the nanopore for detection.

[0159]

[0167] Nucleotide uptake events can be detected in real time by nanopores as they occur. Enzymes such as DNA polymerase attached to or adjacent to the nanopore can facilitate the flow of polynucleotides through or adjacent to the nanopore. In a nucleotide uptake event, i.e., the uptake of multiple nucleotides, one or more tags may be released or presented, which can be detected by the nanopore. Detection can occur as the tags flow through or adjacent to the nanopore, as the tags remain in the nanopore, and / or as the tags are presented to the nanopore. In some cases, enzymes attached to or adjacent to the nanopore may be useful in detecting tags associated with the uptake of one or more nucleotides.

[0160]

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

[0161]

[0169] Nanopores can be formed in a film located adjacent to the sensing electrodes of a sensing circuit, such as an integrated circuit, or embedded therein by other means. 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 the chip or other device having the nanopore, or it may be located off-chip, such as in an off-chip configuration.

[0162]

[0170] As nucleic acids or tags flow through or adjacent to a nanopore, a sensing circuit detects the electrical signals associated with the nucleic acids or tags. Nucleic acids can be subunits of a large chain. Tags can be byproducts of nucleotide incorporation events or other interactions between tagged nucleic acids and the nanopore or species adjacent to the nanopore, such as enzymes that cleave the tags from the nucleic acids. Tags may remain attached to the nucleotides. Detected signals can be collected, stored in a memory location, and later used to construct nucleic acid sequences. Collected signals can be processed to explain any anomalies in the detected signals, such as errors.

[0163]

[0171] Using nanopores, polynucleotides can be sequenced indirectly, and optionally by electrical detection. Indirect sequencing can be any method in which nucleotides incorporated into the growing chain do not pass through the nanopore. Polynucleotides can pass through a range of any suitable distance from the nanopore and / or close to it, optionally within a range in which tags released from nucleotide incorporation events can be detected at the nanopore.

[0164]

[0172] Nanopores can be used to detect byproducts of nucleotide uptake events. Nucleotide uptake events refer to the uptake of nucleotides into a growing polynucleotide chain. Byproducts may correlate with the uptake of a given type of nucleotide. Nucleotide uptake events are catalyzed by enzymes such as DNA polymerase and may use base-pair interactions with a template molecule to select uptake at each position from among the available nucleotides.

[0165]

[0173] Nucleic acid samples can be sequenced using tagged nucleotides or nucleotide analogs. In some examples, a method for sequencing nucleic acid molecules includes (a) incorporating tagged nucleotides (e.g., polymerizing them) such that the tags associated with individual nucleotides are released upon incorporation, and (b) detecting the released tags using a nanopore. In some examples, the method further includes guiding the tags attached to or released from individual nucleotides to pass through a nanopore. The released or attached tags can be detected by any preferred technique. The tag may be guided by an enzyme (or molecular motor) and / or a voltage difference across the pore. Alternatively, the freed or attached tag may be guided through the nanopore without the use of an enzyme. For example, the tag may be guided by a voltage difference across the nanopore, as described herein.

[0166]

[0174] The tag may be detected using a nanopore apparatus having at least one nanopore in the membrane. The tag may associate with the individual tagged nucleotide upon uptake of that individual tagged nucleotide. The nanopore apparatus can detect the tag associated with the individual tagged nucleotide upon uptake. Whether the tagged nucleotide is incorporated into the growing nucleic acid chain or not, it may be detected, determined, or identified by the nanopore apparatus, optionally using the electrodes and / or nanopores of the nanopore apparatus, over a given period of time. The time during which the nanopore apparatus detects the tag may be shorter, and possibly substantially shorter, than the time during which the tag and / or tag-coupled nucleotide is held by an enzyme, such as an enzyme that promotes the uptake of nucleotides into the nucleic acid chain (e.g., polymerase). The tag may be detected by electrodes multiple times over the period during which the incorporated tagged nucleotide is associated with the enzyme. For example, the tag can be detected by an 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 the incorporated tagged nucleotide is associated with the enzyme.

[0167]

[0175] Sequencing can be achieved using pre-loaded tags. Pre-loading a tag may involve guiding at least a portion of the tag through at least a portion of the nanopore while the tag can be attached to a nucleotide and the nucleotide is incorporated into a nucleic acid chain (e.g., a growing nucleic acid chain), while incorporation into the nucleic acid chain is occurring, or while it is not yet incorporated into the nucleic acid chain but incorporation into the nucleic acid chain may occur. Pre-loading a tag may involve guiding at least a portion of the tag through at least a portion of the nanopore before the nucleotide is incorporated into the nucleic acid chain or while the nucleotide is incorporated into the nucleic acid chain. Pre-loading a tag may involve guiding at least a portion of the tag through at least a portion of the nanopore after the nucleotide has been incorporated into the nucleic acid chain.

[0168]

[0176] Tags associated with individual nucleotides can be detected by nanopores without being released from the nucleotides upon incorporation. The tags can also be detected without being released from the incorporated nucleotides during the synthesis of a nucleic acid chain complementary to the target chain. Tags can be attached to nucleotides using linkers so that they are presented to the nanopore (e.g., the tag hangs within or extends through at least a portion of the nanopore). The linker length may be long enough to allow the tag to extend into or through at least a portion of the nanopore. In some examples, the tag is presented to the nanopore (i.e., moves into it) by a voltage difference. Other methods for presenting the tag in the pore may also be preferred (e.g., the use of enzymes, magnets, electric fields, or pressure differences). In some examples, no active force is applied to the tag (i.e., the tag diffuses into the nanopore).

[0169]

[0177] A chip for sequencing nucleic acid samples may include a plurality of individually addressable nanopores. These plurality of individually addressable nanopores may include at least one nanopore formed on a membrane positioned adjacent to an integrated circuit. Each individually addressable nanopore may have the ability to detect a tag associated with a specific 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 in the nanopore in response to a nucleotide uptake event and released from the nucleotide. The released tag can pass through the nanopore. In some cases, the tag does not pass through the nanopore. Tags released in response to a nucleotide uptake event may flow through the nanopore but are distinguished from tags not released in response to a nucleotide uptake event by their residence time in the nanopore, at least in part. In some cases, tags that remain in the nanopore for at least 100 milliseconds (ms) are released in response to a nucleotide uptake event, while tags that remain in the nanopore for less than 100 ms are not released in response to a nucleotide uptake event. The tag may be captured in the nanopore and / or guided through it by a second enzyme or protein (e.g., a nucleic acid-binding protein). The second enzyme may cleave the tag in response to (e.g., during or after) nucleotide uptake. The linker between the tag and the nucleotide may be cleaved.

[0171]

[0179] Tags coupled to incorporated nucleotides are distinguished using nanopores from tags associated with unincorporated nucleotides in the growing complementary chain, based on the residence time of the tags in the nanopore or the signal detected from the unincorporated nucleotides. Unincorporated nucleotides can generate a detectable signal (e.g., voltage difference, current) over a time period of 1 nanosecond (ns) to 100 ms or 1 ns to 50 ms, while incorporated nucleotides can generate a signal with a lifetime of 50 ms to 500 ms or 100 ms to 200 ms. Unincorporated nucleotides can generate a detectable signal over a time period of 1 ns to 10 ms or 1 ns to 1 ms. Unincorporated tags are detectable by nanopores for a longer time (on average) than incorporated tags are detectable by nanopores.

[0172]

[0180] Incorporated nucleic acids can be detected and / or are detectable by nanopores for a shorter time than unincorporated nucleotides. Conversely, incorporated nucleic acids can be detected and / or are detectable by nanopores for a longer time than unincorporated nucleotides. Using these time differences and / or ratios, it is possible to determine whether or not a nucleotide detected by nanopores as described herein has been incorporated.

[0173]

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

[0174]

[0182] A tag or tag species may contain one or more detectable atoms or molecules. A tag may contain one or more adenine, guanine, cytosine, thymine, uracil, or derivatives thereof, linked to any position on a nucleic acid molecule, including phosphate groups, sugars, or nitrogen-containing bases. A tag may contain one or more adenine, guanine, cytosine, thymine, uracil, or derivatives thereof, covalently linked to the phosphate group of a nucleic acid base.

[0175]

[0183] The tags may have a length of at least 0.1 nanometers (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.

[0176]

[0184] The tag may include a tail of repeating subunits, such as multiple adenine, guanine, cytosine, thymine, uracil, or derivatives thereof. For example, the tag may include 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 subunits of adenine, guanine, cytosine, thymine, uracil, or derivatives thereof. The subunits may be linked to each other and at terminal ends linked to the phosphate groups of nucleic acids. Other examples of tag portions include any polymer material, such as polyethylene glycol (PEG), polysulfonates, amino acids, or any fully or partially positively charged, negatively charged, or uncharged polymers.

[0177]

[0185] IV. Polymerase

[0186] DNA polymerase can bind to the 3' end of the strand containing the nicks of polynucleotides at the nicking sites. DNA sequencing can be achieved by amplifying and transcribing polynucleotides in close proximity to nanopores and tagged nucleotides using enzymes such as DNA polymerase. Sequencing methods may involve incorporating or polymerizing tagged nucleotides using polymerases or transcribs such as DNA polymerase. Polymerases can be mutated to enable them to accept tagged nucleotides. Polymerases can also be mutated to increase the time the tags are detected by nanopores.

[0178]

[0187] The sequencing enzyme can be any suitable enzyme that creates polynucleotide chains by phosphate bonding of nucleotides, for example. DNA polymerases include, for example, 9°Nm(trademark) polymerase or its variants, Escherichia coli (E. coli) DNA polymerase I, bacteriophage T4 DNA polymerase, sequencer, and Ta q DNA polymerase, 9°Nm(trademark) polymerase (exo-) A485L / Y40 This may be 9V, Φ29 DNA polymerase, Bst DNA polymerase, or any of the aforementioned variants, mutants, or homologs. The homologs may have any preferred percentage of homology, such as 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, polymerases may be attached to or located adjacent to nanopores for nanopore sequencing. Preferred methods for attaching polymerases to nanopores include crosslinking the enzyme to or adjacent to the nanopore, such as by forming intramolecular disulfide bonds. Nanopores and enzymes may also be fusions, such as those encoded by a single polypeptide chain. Methods for producing fusion proteins may include fusing the enzyme coding sequence with the nanopore coding sequence in-frame and adjacent to it, and expressing this fusion sequence from a single promoter. Polymerases can be attached to or coupled to nanopores using molecular staples or protein fingers. Polymerases can be attached to nanopores via intermediate molecules, such as biotin conjugated to both the enzyme and the nanopore, with streptavidin tetramers linked to both biotin molecules. These intermediate molecules may be called linkers.

[0180]

[0189] Sequencing enzymes can also be attached to nanopores using antibodies. Polymerases can be attached to nanopores using proteins that form covalent bonds between themselves. Phosphatase enzymes or enzymes that cleave tags from nucleotides can also be attached to nanopores.

[0181]

[0190] Polymerases can be mutated to enhance and / or improve the efficiency of tagged nucleotide uptake into growing polynucleotides by mutant polymerases compared to non-mutated polymerases. Polymerases can be mutated to improve the entry of nucleotide analogs, such as tagged nucleotides, into the polymerase's active site region, and / or to cooperate with nucleotide analogs in the active site.

[0182]

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

[0183]

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

[0184]

[0193] The polymerase can be adapted to enable the detection of sequencing events. The polymerase rate profile may 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 polymerase rate profile may be such that the tag is loaded into the nanopore 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 may 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 may be configured to enable the detection of one or more events related to the amplification and transcription of cyclic polynucleotides. These one or more events may be kinetically observable and / or non-kinetically observable, such as nucleotides moving through the nanopore without contact with the polymerase.

[0186]

[0195] In some cases, a polymerase reaction exhibits two kinetic steps: one starting from an intermediate in which the nucleotide or polyphosphate product is bound to the polymerase enzyme, and another starting 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] A suitable polymerase may exhibit potent or enhanced chain displacement.

[0188]

[0197] V. Identification of sequence variants

[0198] The methods provided herein can be used to identify sequence variants in polynucleotide samples. A sequence difference between a sequencing read and a reference sequence is referred to as a genuine sequence variant if the sequence difference appears in at least two different polynucleotides, for example, two different cyclic polynucleotides, and can be distinguished as a result of having different junctions. Since the location and type of a sequence variant resulting from amplification or sequencing errors are unlikely to be exactly repeated in two different polynucleotides containing the same target sequence, including this validation parameter can reduce the background of false sequence variants and, at the same time, increase the sensitivity and accuracy of detecting actual sequence mutations in the sample. Sequence variants can occur at frequencies of less than or equal to 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%, and 0.001%, which are statistically significant above the background for accurate identification to be possible. Sequence variants can occur at frequencies of less than 0.1%. The frequency of a sequence variant can be statistically significant above the background if such a frequency has a p-value less than, for example, 0.05, 0.01, 0.001, or 0.0001, and is statistically significant above the background error rate. The frequency of a sequence variant may be considered well above the background if its frequency is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 100 times or more than the background error rate. The background error rate for accurately determining the 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 involve optimally aligning one or more sequencing reads with a reference sequence to identify the differences between the two, and identifying junctions. Alignment may involve placing one sequence along another, introducing repeating gaps along each sequence, scoring how well the two sequences match, and repeating this for various positions along the reference. The best-scoring match is considered the alignment, which corresponds to an inference about the degree of relationship between the sequences.

[0190]

[0200] The reference sequence used to compare a sequencing read is a reference genome, such as the genome of a member of the same species as the target. The reference genome may be complete or incomplete. The reference genome may consist only of regions containing the target polynucleotide, such as those derived from the reference genome or from a consensus generated from the sequencing read under analysis. The reference sequence may contain or consist of polynucleotide sequences from one or more organisms, such as those from one or more bacteria, archaea, viruses, protists, fungi, or other organisms. The reference sequence may consist only of 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 may be the entire genome of a pathogen, such as a specific strain or serotype of the pathogen, or a portion of it useful for identification. Sequencing reads can be aligned with multiple different reference sequences, such as for screening against 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 characterizing patient samples and, optionally, diagnosing a condition in question. Therapeutic applications include providing information based on the results of the methods provided herein regarding the selection of therapies in which the patient's response may be best and / or the treatment of a subject requiring therapeutic intervention. This may include.

[0192]

[0203] For example, the methods provided herein can be used to diagnose the presence of a tumor, its progression, and / or metastasis, such as when the polynucleotide under analysis contains or consists of cfDNA, ctDNA, or fragmented tumor DNA. The subject may be monitored for the effectiveness of tumor treatment, for example, by monitoring ctDNA over time, with a decrease in ctDNA being used as an indicator of treatment effectiveness, and an increase in ctDNA providing information regarding the selection of different treatments and / or different dosages. Other uses include the evaluation of organ rejection in transplant recipients, such as when an increase in circulating DNA volume corresponding to the transplant donor genome is used as an early indicator of graft rejection, and gene typing / isotyping of pathogen infections, such as viral or bacterial infections. Detection of sequence variants in circulating fetal DNA may be used to diagnose fetal pathology.

[0193]

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

[0194]

[0205] The causative genetic variants may include sequence variants associated with a specific type or stage of cancer, or sequence variants of cancers with specific characteristics, such as metastatic potential, drug resistance, and / or drug responsiveness. The methods provided in this disclosure can provide information regarding treatment decisions, guidance, and monitoring of cancer therapy. For example, treatment effectiveness can be monitored by comparing pre-treatment, intra-treatment, and post-treatment patient ctDNA samples, particularly those involving molecularly targeted therapies such as monoclonal drugs, chemotherapy agents, radiotherapy protocols, and any combination thereof. For example, ctDNA can be monitored to determine whether a particular mutation increases or decreases after treatment, or whether a new mutation appears, which may allow physicians to modify treatment in a much shorter time than would be possible with monitoring methods that track the patient's condition. The methods may include diagnosing a subject based on the results of polynucleotide sequencing, such as diagnosing the subject as having a specific stage or type of cancer associated with the detected sequence variant, or reporting the likelihood that a patient has or will develop such cancer.

[0195]

[0206] For example, with therapies specifically targeted to patients based on molecular markers, patients can be examined to find out if specific mutations are present in their tumors, and these mutations can be used to predict the response to or resistance to the therapy, guiding the decision of whether or not to use that therapy. Detection and monitoring of ctDNA during the course of treatment may be useful in guiding treatment selection.

[0196]

[0207] One or more cancer-related sequence variants may be used for diagnosis, prognosis, or treatment decisions. For example, suitable oncologically meaningful target sequences include modifications of the TP53, ALK, KRAS, PIK3CA, BRAF, EGFR, and KIT genes. Target sequences that can be specifically amplified and / or specifically analyzed with respect to a sequence variant may be all or some cancer-related genes.

[0197]

[0208] The methods provided by this disclosure may 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 the characteristics under analysis, such as a specific disease, cancer type, and / or cancer stage, the methods provided by this disclosure can be used to identify sequence variants that reflect mutations in a specific gene or gene part, compared to individuals that do not possess that characteristic. For identified sequence variants that appear at a statistically significant frequency within a population, a degree of association with a particular characteristic can be assigned. Then, the identified sequence variant or type of sequence variant can be used for the diagnosis or treatment of individuals found to possess it.

[0198]

[0209] Further therapeutic applications include use in non-invasive fetal diagnosis. Fetal DNA may be present in the blood of a pregnant woman. The methods provided herein allow for the identification of sequence variants in circulating fetal DNA, and thus for the diagnosis of one or more hereditary disorders of the fetus, such as hereditary 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 to be used for comparison. The control sample may be any suitable tissue, which can then be sequenced to provide a reference sequence. The sequence of cfDNA corresponding to the fetal genomic DNA can then be identified as a sequence variant compared to the maternal reference. The father may also provide a reference sample that is useful for identifying fetal sequences and sequence variants.

[0199]

[0210] Other therapeutic applications may include the detection of exogenous polynucleotides, including those from pathogens such as bacteria, viruses, fungi, and microorganisms, and this information may inform treatment options.

[0200]

[0211] VII. Computer Systems

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

[0201]

[0213] Figure 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 may communicate, for example, with 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 detection. The computer system 601 may also communicate with one or more controllers or processors of the present disclosure. The computer system 601 may be a computer system remotely installed on or from a user's electronic device. The electronic device may be a mobile electronic device.

[0202]

[0214] Computer system 601 includes a central processing unit (CPU, also herein referred to as "processor" and "computer processor") 605, which may be a single-core or multi-core processor or multiple processors for parallel processing. Computer system 601 also includes memory or storage locations 610 (e.g., random-access memory, read-only memory, flash memory), electronic storage devices 615 (e.g., hard disks), communication interfaces 620 for communicating with one or more other systems (e.g., network adapters), and peripheral devices 625, such as caches, other memory, data storage, and / or electronic display adapters. The memory 610, storage devices 615, interfaces 620, and peripheral devices 625 communicate with the CPU 605 through a communication bus (solid line), such as a motherboard. Storage devices 615 may be data storage devices (or data repositories) for storing data. Computer system 601 may be operably coupled to a computer network ("network") 630 using the communication interface 620. Network 630 may be the Internet, the Internet and / or an extranet, or an intranet and / or extranet communicating with the Internet. Network 630 may also, depending on the circumstances, communicate remotely and It is a and / or 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 using computer system 601, which may allow devices coupled to computer system 601 to act as clients or servers.

[0203]

[0215] The CPU 605 can execute a series of machine-readable instructions, which may be embodied as a program or software. Instructions may be stored in a memory location such as memory 610. Instructions may be directed to the CPU 605, which may then be programmed or otherwise configured to implement the methods of this disclosure. Examples of operations performed by the CPU 605 include retrieval, decryption, execution, and writing.

[0204]

[0216] CPU 605 may be part of a circuit, such as an integrated circuit. The circuit may include one or more other components of 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 also store user data, such as user settings and user programs. The computer system 601 may include one or more additional data storage devices that are external to the computer system 601, such as those located on remote servers communicating with the computer system 601 via 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 user's remote computer system. Examples of remote computer systems include personal computers (e.g., portable PCs), slate or tablet PCs (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, smartphones (e.g., Apple® iPhone, Android devices, Blackberry®), or personal digital assistants. Users can access computer system 601 via network 630.

[0207]

[0219] The methods described herein may be implemented using machine-executable code (e.g., a computer processor) stored in an electronic storage location of the computer system 601, such as memory 610 or electronic storage device 615. The machine-executable or machine-readable code may be provided in the form of software. When in use, the code may be executed by the processor 605. In some cases, the code may be retrieved from storage device 615 and stored in memory 610 to facilitate access by the processor 605. In some circumstances, electronic storage device 615 may be excluded, and machine-executable instructions may be stored in memory 610.

[0208]

[0220] The code may be pre-compiled and configured for use with a machine that has a processor adapted to run the code, or it may be compiled at runtime. The code may be supplied in a programming language, which may be selected to allow execution of the code in a pre-compiled form or in its compiled-as-is form.

[0209]

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

[0210]

[0222] Therefore, machine-readable media, such as computer-executable code, can take many forms, including but not limited to tangible storage media, carrier media, or physical transmission media. Non-volatile storage media include optical disks or magnetic disks, such as any one or more storage devices in any computer, which may be used in the implementation of databases, etc., as shown in the drawings. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wires and optical fibers, including wires, including buses in computer systems. Carrier media can take the form of electrical or electromagnetic signals, or sound waves or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Therefore, common forms of computer-readable media include, for example, floppy disks, flexible disks, hard disks, magnetic tapes, any other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punch cards, paper tapes, any other physical storage media having perforated patterns, RAM, ROMs, PROMs and EPROMs, FLASH-EPROMs, any other memory chips or cartridges, carrier data or instructions, cables or links that carry such carriers, or any other media from which a computer can read programming code and / or data. Many of these forms of computer-readable media can be involved in transporting one or more sequences of one or more instructions to a processor for execution.

[0211]

[0223] The computer system 601 may include, or communicate with, an electronic display 635 including a user interface (UI) 640 for providing, for example, (i) the progress of the reaction mixture, (ii) the progress of 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 this disclosure may be implemented using one or more algorithms. The algorithms may be implemented using software at runtime by the central processing unit 605. The algorithms may, for example, target nucleotides, polynucleotides, peptides, and This technology can determine the sequence readout of lipeptides, proteins, and other molecules.

[0213]

[0225] While preferred embodiments of the present invention are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided merely as examples. The present invention is not intended to be limited by the specific examples provided herein. Although the present invention is described with reference to the foregoing specification, the descriptions and examples of embodiments herein are not intended to be constrained. Herein, those skilled in the art will be able to conceive of numerous variations, alterations, and substitutions without departing from the present invention. Furthermore, it should be understood that none of the embodiments of the present invention are limited to the specific descriptions, configurations, or relative proportions shown herein, and that they depend on various conditions and variables. It should be understood that in the practice of the present invention, various alternative forms of the embodiments of the present invention described herein may be utilized. Therefore, the present invention is intended to encompass any such alternative forms, improvements, variations, or equivalents. The following claims define the scope of the present invention, and methods and structures within the scope of these claims and their equivalents are intended to be included within the scope of the present invention.

Claims

1. A system for analyzing or identifying target molecules, A sensor comprising: (i) a sensing electrode; (ii) a bonding unit coupled to the sensing electrode and configured to bond 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 the surface of the sensing electrode. Equipped with, The sensor is configured to detect one or more signals indicating the impedance or impedance change of the sensor when at least a portion of the target molecule is bound by the binding unit, and the one or more signals are available for analysis or identification of the target molecule. system.

2. The system according to claim 1, wherein one or more signals indicate (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 according to claim 1, wherein the one or more signals are current or voltage.

4. The system according to claim 3, wherein one or more of the signals are not tunnel currents.

5. The system according to claim 1, wherein the average cross-sectional dimensions of the sensing electrodes are 20 times or less the average size of the target molecule.

6. The system according to claim 5, wherein the average cross-sectional dimension of the sensing electrode is less than or equal to twice the average size of the target molecule.

7. The system according to claim 1, wherein the average cross-sectional dimensions of the sensing electrodes are smaller than the average size of the target molecules.

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

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

10. The system according to claim 1, wherein the target molecule includes a tag, and the tag is configured to induce a change in one or more signals.

11. The system according to claim 1, wherein the sensor further includes a reference electrode electrically in communication with the sensing electrode, and the one or more signals indicate the impedance or impedance change between the sensing electrode and the reference electrode.

12. The system according to claim 11, wherein 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 field generator configured to apply a second electric field along a second direction different from the first direction.

13. The system according to claim 12, wherein the second direction is substantially orthogonal to the first direction.

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

15. The system according to claim 1, wherein the binding unit comprises one or more members selected from the group consisting of small molecules, enzymes, antibodies, functional fragments thereof, and functional variants thereof.

16. The system according to 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 their variants.

17. A method for analyzing or identifying a target molecule, (a) (i) a sensing electrode; (ii) a bonding unit coupled to the sensing electrode and configured to bond 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 the surface of the sensing electrode; (b) detecting one or more signals indicating the impedance or impedance change of the sensor when at least the portion of the target molecule is bound by the binding unit; and (c) Analyzing or identifying the target molecule using one or more of the signals. A method that includes this.

18. The method according to claim 17, wherein one or more signals indicate (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 according to claim 17, wherein the one or more signals are current or voltage.

20. The method according to claim 19, wherein one or more of the signals are not tunnel currents.

21. The method according to claim 17, wherein the average cross-sectional dimensions of the sensing electrode are 20 times or less the average size of the target molecule.

22. The method according to claim 21, wherein the average cross-sectional dimensions of the sensing electrode are less than or equal to twice the average size of the target molecule.

23. The method according to claim 17, wherein the average cross-sectional dimensions of the sensing electrode are smaller than the average size of the target molecule.

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

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

26. The method according to claim 17, wherein the target molecule includes a tag, and the tag is configured to induce a change in one or more signals.

27. The method according to claim 17, wherein the sensor further includes a reference electrode electrically in communication with the sensing electrode, and the one or more signals indicate the impedance or impedance change between the sensing electrode and the reference electrode.

28. The method according to claim 27, wherein the sensing electrode and the reference electrode provide a first electric field along a first direction, and the method further comprises using an additional field generator to apply a second electric field along a second direction different from the first direction.

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

30. The method according to claim 17, further comprising determining the residence time of at least the portion of the target molecule on the bond unit.

31. The method according to claim 17, wherein the binding unit comprises one or more members selected from the group consisting of small molecules, enzymes, antibodies, functional fragments thereof, and functional variants thereof.

32. The method according to 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 their variants.

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